Methods of producing tissue-derived epithelial organoids and uses thereof

Abstract

The present disclosure provides methods of making tissue-derived epithelial organoids. In particular, the present disclosure provides a tissue-derived epithelial organoid embedded in a hydrogel suspended in a culture medium. The present disclosure further provides methods of using such organoids.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 508,132, filed June 14, 2023, the contents of which are incorporated by reference in their entirety. TECHNICAL FIELD

[0002] The subject matter disclosed herein relates to tissue-derived epithelial organoids and methods of generating and using such organoids. BACKGROUND

[0003] Tissue-derived epithelial organoids are three-dimensional (3D) multicellular spheroids that recapitulate the complexity and function of tissues in vivo and have become physiologically relevant in vitro models of tissues. For example, intestinal organoids, also known as “enteroids” or “colonoids,” are derived from adult stem cells isolated from primary intestinal tissue and can be easily propagated and cryopreserved for long-term storage. Intestinal organoids can differentiate into various intestinal cell types, perform epithelial functions such as barrier maintenance, absorption, secretion, and digestion, and recapitulate the biological characteristics and clinical responses of the patient from which these intestinal organoids are derived (Clevers (2016) Cell 165, 1586-1597; Zachos et al. (2016) J Biol Chem 291, 3759-3766). As a result, many intestinal organoids have been widely adopted, replacing traditional transformed and immortalized intestinal cell lines, to generate fundamental scientific discoveries and facilitate translational applications in numerous areas including cancer biology, infectious diseases, and cystic fibrosis (Clevers (2016); Schutgens and Clevers (2019) Annu Rev Pathology Mech Dis 15, 1-24).

[0004] Challenges to implementation of tissue-derived epithelial organoids in fields such as drug development lie in the difficulty of scaling existing organoid culture techniques, which require tedious manual methods or development of advanced automation infrastructure (Louey et al. (2021) Slas Discov 26, 1138-1147). In existing techniques, tissue-derived epithelial stem cells (isolated from primary tissue or passaged from established organoid cultures) are resuspended in a cold extracellular matrix (ECM) solution, most commonly a CULTREX® basement membrane extract (BME) or MATRIGEL® hydrogel. The ECM is deposited onto the surface of a plate, then warmed to solidify the ECM cell solution into a surface-attached hydrogel dome, and covered with culture media. The media in each well is changed every few days, and organoids form over a period of 1 to 2 weeks (Mahe et al. Curr Protoc Mouse Biology 3, 217-240; Pleguezuelos-Manzano et al. (2020) Curr Protoc Immunol 130, e106; Sato et al. (2009) Nature 459, 262-265; Sato et al. (2011) Gastroenterology 141, 1762-1772). This technique is difficult to scale, as it is limited by the available surface area for hydrogel dome formation, is time- and labor-intensive, and is prone to user handling errors, and diffusion limitations of the hydrogel lead to organoid growth and morphological heterogeneity (Park et al. (2022) Nat Methods 19, 1449-1460; Ringel et al. (2020) Cell Stem Cell 26, 431-440.e8; Shin et al. (2020) iScience 23, 101372). Thus, there is a need in the art for more efficient and high-throughput methods of producing tissue-derived epithelial organoids. SUMMARY

[0005] The subject matter disclosed herein relates to tissue-derived epithelial organoids and methods of producing such organoids. The present disclosure further provides methods of using tissue-derived epithelial organoids and systems for performing the methods disclosed herein.

[0006] In certain embodiments, a method of generating a tissue-derived epithelial organoid comprises (a) contacting tissue-derived epithelial stem cells with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture, (b) suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture, and (c) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to generate a tissue-derived epithelial organoid. In certain embodiments, a plurality of tissue-derived epithelial stem cells are contacted with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the method further comprises fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate a fragmented structure comprising a tissue-derived epithelial organoid.

[0007] In certain embodiments, the hydrogel solidifies upon contact with the culture medium. In certain embodiments, suspending the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium comprises submerging a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the culture medium. In certain embodiments, the temperature of the culture medium is from about 25 °C to about 50 °C. In certain embodiments, the temperature of the culture medium is from about 30 °C to about 50 °C. In certain embodiments, the temperature of the culture medium is from about 30 °C to about 40 °C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is less than about 20 °C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2 °C to about 25 °C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2 °C to about 20 °C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2 °C to about 10 °C.

[0008] The present disclosure further provides a method of generating a suspension culture of tissue-derived epithelial organoids. In certain embodiments, the method comprises (a) introducing a mixture comprising a hydrogel and tissue-derived epithelial stem cells into a culture medium to generate a suspended mixture; and (b) culturing the suspended mixture in the culture medium to generate tissue-derived epithelial organoids in suspension. In certain embodiments, the mixture introduced into the culture medium comprises a hydrogel and a plurality of tissue-derived epithelial stem cells. In certain embodiments, the method comprises (a) introducing a mixture comprising a hydrogel and a plurality of tissue-derived epithelial stem cells into a culture medium to generate a suspended mixture, and (b) culturing the mixture in the culture medium to generate tissue-derived epithelial organoids in suspension. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. In certain embodiments, introducing the mixture into the culture medium comprises submerging a dispensing device containing the mixture in the culture medium and dispensing the mixture into the culture medium. In certain embodiments, the temperature of the culture medium is from about 25 °C to about 50 °C. In certain embodiments, the temperature of the culture medium is from about 30 °C to about 50 °C. In certain embodiments, the temperature of the culture medium is from about 25 °C to about 40 °C. In certain embodiments, the temperature of the culture medium is from about 30 °C to about 40 °C. In certain embodiments, the temperature of the mixture is less than about 20 °C. In certain embodiments, the temperature of the mixture is from about 2 °C to about 25 °C. In certain embodiments, the temperature of the mixture is from about 2 °C to about 20 °C. In certain embodiments, the temperature of the mixture is from about 2 °C to about 10 °C. In certain embodiments, the method further comprises fragmenting the suspended mixture to generate fragmented structures comprising tissue-derived epithelial organoids.

[0009] In certain embodiments, a method for producing a tissue-derived epithelial organoid in suspension culture comprises (a) contacting a tissue-derived epithelial stem cell with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture; (b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate; (c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to produce a solidified hydrogel-tissue-derived epithelial stem cell mixture; (d) suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture; and (e) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in culture medium to produce a tissue-derived epithelial organoid. In certain embodiments, a plurality of tissue-derived epithelial stem cells is contacted with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. For example, and without limitation, a method comprises (a) contacting a plurality of tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture, (b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate, (c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to produce a solidified hydrogel-tissue-derived epithelial stem cell mixture, (d) suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture, and (e) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in culture medium to produce a tissue-derived epithelial organoid. In certain embodiments, the method further comprises removing the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate prior to suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in culture medium.

[0010] In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate as a droplet. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate to have a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the method further comprises fragmenting the hydrogel-tissue-derived epithelial stem cell mixture in culture medium to produce a fragmented structure comprising a tissue-derived epithelial organoid.

[0011] In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture, the suspended mixture, or the solidified hydrogel-tissue derived epithelial stem cell mixture comprises a length, width, and / or diameter of greater than about 0.1 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture, the suspended mixture, or the solidified hydrogel-tissue derived epithelial stem cell mixture comprises a length, width, and / or diameter of about 0.1 mm to about 1,000 mm (e.g., about 0.1 mm to about 20 mm). In certain embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture, the suspended mixture, or the solidified hydrogel-tissue derived epithelial stem cell mixture is in the form of a droplet. In certain embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture, the suspended mixture, or the solidified hydrogel-tissue derived epithelial stem cell mixture has a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape.

[0012] In certain embodiments, the tissue derived epithelial stem cell or plurality of tissue derived epithelial stem cells is comprised within a tissue fragment, an organoid fragment, or a combination thereof. In certain embodiments, the tissue derived epithelial stem cell or plurality of tissue derived epithelial stem cells is isolated from a primary epithelial tissue. In certain embodiments, the tissue derived epithelial stem cell is obtained from a fragment of a tissue selected from the group consisting of a lacrimal gland, a tonsil, a salivary gland, a gastrointestinal tissue, a thyroid, a lung, a breast, a liver, a bile duct, a stomach, a kidney, a pancreas, an endometrium, a fallopian tube, a cervix, a prostate, a bladder, an ovary, a taste bud, a placenta, and combinations thereof. Alternatively or additionally, the tissue derived epithelial stem cell is obtained from a fragment of an organoid selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a breast organoid, a liver organoid, a bile duct organoid, a stomach organoid, a kidney organoid, a pancreas organoid, an endometrium organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovary organoid, a taste bud organoid, a cytotrophoblast organoid, and combinations thereof.

[0013] In certain embodiments, the plurality of tissue derived epithelial stem cells comprises about 1 x 10 4 tissue derived epithelial stem cells per ml of hydrogel to about 1 x 10 7 tissue derived epithelial stem cells per ml of hydrogel.

[0014] In certain embodiments, the hydrogel is selected from the group consisting of a synthetic hydrogel, a natural hydrogel, and combinations thereof. In certain embodiments, the natural hydrogel comprises basement membrane extract (BME) or extracellular matrix (ECM) components. In certain embodiments, the hydrogel has a protein concentration greater than about 1 mg / ml. In certain embodiments, the hydrogel comprises greater than about 1 w / v % of a BME component, an ECM component, or a polymer, in w / v %. In certain embodiments, the hydrogel has a storage modulus G' that is equal to or greater than a loss modulus G''.

[0015] In certain embodiments, the culture medium is present in a container. In certain embodiments, the container is a culture dish, a multi-well plate, a conical tube, a receptacle, a culture bag, a bioreactor, or a flask.

[0016] The present disclosure further provides a tissue-derived epithelial organoid produced by the methods disclosed herein.

[0017] In certain embodiments, the tissue-derived epithelial organoid produced by the methods of the present disclosure has a uniform morphology compared to a reference tissue-derived epithelial organoid. In certain embodiments, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a substrate. In certain embodiments, the tissue-derived epithelial organoid has a uniform size. In certain embodiments, the tissue-derived epithelial organoid has a more uniform average diameter than the reference tissue-derived epithelial organoid.

[0018] In certain embodiments, a stem cell and / or a proliferation marker is expressed at a higher level in the population of tissue-derived epithelial organoids produced by the methods of the present disclosure compared to a reference population of tissue-derived epithelial organoids. In certain embodiments, a differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids produced by the methods of the present disclosure compared to a reference population of tissue-derived epithelial organoids. In certain embodiments, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a substrate. In certain embodiments, the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In certain embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

[0019] The present disclosure further provides a composition comprising a tissue-derived epithelial organoid and a culture medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the culture medium. In certain embodiments, the geometry of the hydrogel comprises a length, width, and / or diameter of greater than about 0.1 mm. In certain embodiments, the geometry of the hydrogel comprises a length, width, and / or diameter of about 0.1 mm to about 1,000 mm (e.g., about 0.1 mm to about 20 mm). In certain embodiments, the hydrogel is a droplet. In certain embodiments, the hydrogel has a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, a stem cell and / or a proliferation marker is expressed at a higher level in the population of tissue-derived epithelial organoids compared to a reference population of tissue-derived epithelial organoids. In certain embodiments, the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In certain embodiments, a differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids compared to a reference population of tissue-derived epithelial organoids. In certain embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In certain embodiments, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a substrate.

[0020] The present disclosure further provides a method for screening an agent (e.g., a therapeutic agent). In certain embodiments, the method comprises (a) contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids or a composition of tissue-derived epithelial organoids with an agent (e.g., a therapeutic agent), and (b) analyzing the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for a change indicative of effectiveness, disposition, and / or toxicity of the agent (e.g., a therapeutic agent). In certain embodiments, the agent (e.g., a therapeutic agent) is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for about 1 minute to about 3 years, (e.g., about 15 minutes to about 3 years). In certain embodiments, the agent is a therapeutic agent. In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell-based therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. In certain embodiments, the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of a cell signaling pathway, inhibition of a cell signaling pathway, enzyme activity, barrier integrity, and a combination thereof.

[0021] The present disclosure further provides a method of performing a genomic screen. In certain embodiments, the method comprises (a) providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids or a composition of tissue-derived epithelial organoids, (b) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid, and (c) analyzing a change in the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids associated with the mutation. In certain embodiments, the mutation is generated using a gene regulation system. In certain embodiments, the gene regulation system is a gene editing system. In certain embodiments, the gene editing system is a CRISPR system. In certain embodiments, the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of a cell signaling pathway, inhibition of a cell signaling pathway, enzyme activity, barrier integrity, and a combination thereof.

[0022] The present disclosure further provides a method of generating an epithelial cell model. In certain embodiments, the method comprises (a) providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids or a composition of tissue-derived epithelial organoids, (b) digesting the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids into single cells, and (c) culturing the single cells in a culture medium to generate a cell monolayer. In certain embodiments, the single cells are cultured on a permeable cell culture insert. In certain embodiments, the culture medium is a differentiation culture medium. In certain embodiments, the culture medium is a cell growth culture medium. In certain embodiments, the culture medium is a stem cell promoting culture medium.

[0023] The present disclosure further provides a screening method using a cell monolayer generated by the methods described herein. In certain embodiments, the screening method is a method for screening an agent (e.g., a therapeutic agent). For example, but not by way of limitation, the method can comprise (a) contacting a cell monolayer generated by the methods described herein with an agent (e.g., a therapeutic agent); and (b) analyzing the cell monolayer for changes indicative of effectiveness, disposition, and / or toxicity of the agent (e.g., a therapeutic agent). In certain embodiments, the agent (e.g., a therapeutic agent) is contacted with the cell monolayer for about 1 minute to about 3 years, (e.g., about 15 minutes to about 3 years). In certain embodiments, the agent is a therapeutic agent. In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell-based therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. In certain embodiments, the screening method is a genomic screen. In certain embodiments, the method of performing a genomic screen can comprise (a) providing a cell monolayer generated by the methods as described herein; (b) generating a mutation in the genome of one or more cells of the cell monolayer; and (c) analyzing the cell monolayer for changes associated with the mutation. In certain embodiments, the mutation is generated using a gene regulation system. In certain embodiments, the gene regulation system is a gene editing system. In certain embodiments, the gene editing system is a CRISPR system. In certain embodiments, the changes are changes in properties selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of a cell signaling pathway, inhibition of a cell signaling pathway, enzyme activity, barrier integrity, and combinations thereof.

[0024] In certain embodiments, one or more steps of the methods of the present disclosure can be performed using one or more robots and / or automated components. For example, and without limitation, one or more steps of the methods of generating tissue-derived epithelial organoids as described herein, the methods of generating a suspension culture of tissue-derived epithelial organoids, the methods of screening for pharmaceutical agents, the methods of performing genomic screens, the methods of generating epithelial cell models, and / or the screening methods using cell monolayers can be performed using one or more robots and / or automated components. In certain embodiments, the one or more robots and / or automated components are selected from the group consisting of a liquid handling robot, a 3D printer, a syringe pump, and combinations thereof. In certain embodiments, the one or more robots and / or automated components comprise a liquid handling robot.

[0025] The present disclosure further provides systems for culturing tissue-derived epithelial organoids. In certain embodiments, the system comprises a tissue-derived epithelial organoid and a culture medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the culture medium. In certain embodiments, the hydrogel has a geometry comprising a length, width, and / or diameter greater than about 0.1 mm. In certain embodiments, the hydrogel has a geometry comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm (e.g., about 0.1 mm to about 20 mm). In certain embodiments, the hydrogel is a droplet. In certain embodiments, the hydrogel has a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, a stem cell and / or a proliferation marker is expressed at a higher level in the population of tissue-derived epithelial organoids compared to a reference population of tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a substrate. In certain embodiments, the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In certain embodiments, a differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids compared to a reference population of tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a substrate. In certain embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In certain embodiments, the tissue-derived epithelial organoid is selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a breast organoid, a liver organoid, a bile duct organoid, a stomach organoid, a kidney organoid, a pancreas organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovary organoid, a taste bud organoid, a cytotrophoblast organoid, and combinations thereof.In certain embodiments, the system comprises one or more robots and / or automated components for producing and / or culturing the tissue-derived epithelial organoid. In certain embodiments, the one or more robots and / or automated components are selected from the group consisting of a liquid handling robot, a 3D printer, a syringe pump, and combinations thereof. In certain embodiments, the one or more robots and / or automated components comprise a liquid handling robot.

[0026] The present disclosure further provides systems for performing the methods disclosed herein. For example, and without limitation, the present disclosure provides systems for performing the methods of producing a tissue-derived epithelial organoid, the methods of producing a suspension culture of a tissue-derived epithelial organoid, the methods of screening a pharmaceutical agent, the methods of performing a genomic screen, the methods of producing an epithelial cell model, and / or the screening methods using a cell monolayer. In certain embodiments, the system comprises one or more robots and / or automated components for producing and / or culturing the tissue-derived epithelial organoid. In certain embodiments, the one or more robots and / or automated components are selected from the group consisting of a liquid handling robot, a 3D printer, a syringe pump, and combinations thereof. In certain embodiments, the one or more robots and / or automated components comprise a liquid handling robot. BRIEF DESCRIPTION OF DRAWINGS

[0027] FIGS. 1A-1H show the scaling of intestinal organoids in suspension BME hydrogel. (A) Schematic, (B) photograph, and (C) brightfield microscopy of human colon organoids in conventional dome culture (top) and suspended BOBA (organoid bead assembly embedded in BME) culture (bottom). Scale bar for micrographs is 500 pm. (D) Organoid diameter in dome and BOBA cultures in brightfield images. (E) Percentage of Ki67 positive cells in confocal images. Data presented are mean ± SD, Student’s t-test, n = 3, 10 fields each. (F) 3D-reconstructed confocal images of colon organoids in dome or BOBA culture. Ki67 is green, nuclei are blue, and actin is white, and scale bar is 20 pm. (G, H) (G) Colon and (H) ileum organoids total viable cells, cells per cm2 surface area, or cells per pL of BME. All data presented are mean ± SD, Student’s t-test, *p < 0.05, ****p < 0.0001, n = 3 experiments. 2 Table surface area, or cells per pL of BME. All data presented are mean ± SD, Student’s t-test, *p < 0.05, ****p < 0.0001, n = 3 experiments.

[0028] FIGS. 2A-2C show organoid differentiation in BOBA cultures. (A) Brightfield images of colon organoids in dome (top) and BOBA (bottom) cultures show comparable morphology of proliferating and differentiating organoids. Scale bar is 100 pm. (B) Bulk RNA-seq shows that colon organoids cultured in domes and BOBAs similarly downregulate stem and progenitor cell markers and upregulate differentiation markers upon transition from growth to differentiation media. (C) Markers for differentiated epithelial cell types (MUC2 for goblet cells, FABP1 for enterocytes, and CHGA for enteroendocrine cells) are expressed in organoids cultured in both dome (top) and BOBA (bottom) formats. Nuclei are blue, actin is white, and scale bar is 10 pm.

[0029] FIGS. 3A-3C show that BME volume and culture vessel can influence organoid growth in suspended BME hydrogel cultures. (A) Brightfield images of colon organoids in surface-attached domes (50 pL BME in 0.5 mL media) in 24-well plates, or in BOBA cultures (0.5, 1, or 2 mL BME in 5 mL media) in 6-well plate wells. Scale bar is 200 pm. (B, C) (B) 6-well plates or (C) 25 cm 2 organoid diameter, total live cells, live cells per cm 2 surface area, and live cells per pL BME were quantified. Data presented are mean ± SD, one-way ANOVA with multiple comparison test, n = 3 experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0030] FIGS. 4A-4E show organoid size uniformity in BOBA cultures. (A) Schematic depicting imaging strategy for uniformity analysis. (B, C) Brightfield images of colonic organoids in 50 pl BME domes or 10 pl of suspended BOBA hydrogel at the deepest plane. Scale bars are (B) 200 pm and (C) 1 mm. (D) Quantification of organoid diameter in horizontal ROIs of domes or BOBA hydrogel as a function of position across the X-axis. Data presented are mean ± SD, and n = 3 replicates in representative experiments of 3 experiments. (E) Quantification of mean organoid diameter at the edge or core of dome and BOBA cultures. All data presented are mean ± SD, two-way ANOVA, and n = 3 replicates in representative experiments of 3 experiments.

[0031] FIGS. 5A-5B show gene expression of organoids in dome and BOBA cultures. (A, B) Bulk RNA-seq analysis shows differences in gene expression of (A) stem cell and proliferation markers and (B) intestinal epithelial cell markers between colonic organoids in dome cultures or suspended BOBA cultures after 7 days of culture in growth media. Data presented are mean ± SD of normalized counts determined by DESeq2, n = 3 replicates, and statistical analysis by negative binomial model with BH adjusted p-values, *paj < 0.05, **paj < 0.01.

[0032] FIGS. 6A-6D show comparable organoid growth in alternative suspended BME hydrogel culture formats. (A) Photos and (B) brightfield images of colonic organoids in 6-well plate cultures in BOBA, SOBA (syringe-extruded organoid BME components), or SOBA fragments. Alternative formats enable accelerated culture preparation for scale-up. Scale bar is 1 mm. (C) Quantification of organoid diameter and (D) viable cells per well. Data presented are mean ± SD, one-way ANOVA multiple comparison test, n = 3 experiments.

[0033] FIGS. 7A-7E show application of suspended BME hydrogel organoid cultures in medium-throughput screening. (A) Experimental schematic. 225 cm2of organoids were seeded into 96-well plates in 50 pl of BME or BOBA hydrogel. 2 SOBA fragment organoid cultures in flasks were ground to achieve a uniform organoid suspension and then seeded into 96-well plates. (B) Brightfield images of organoids in 225 cm2of BME or BOBA hydrogel in 96-well plates. Scale bar is 1 mm. (C) Quantification of organoid diameter in 96-well plates. Data presented are mean ± SD, one-way ANOVA multiple comparison test, n = 3 experiments. 2Brightfield images of SOBA fragment cultures grown in flasks. Scale bar is 1 mm. (C) Brightfield images of randomly selected wells across a 96-well plate showing comparable inter-well organoid density. Scale bar is 1 mm. (D) Cell Titer Glo 3D (CTG) viability readouts showing similar inter-well variability between domes and suspended BME cultures in a 96-well plate. Data presented are mean ± SD. Student's t-test. (E) Representative dose response viability curves (CTG assay) of suspended BME organoids treated with diacerein, sorafenib, SN-38, or docetaxel for 3 days. Data presented are mean ± SD, n = 4.

[0034] FIGS. 8A-8C show the use of suspended BME hydrogel organoids to generate Transwell monolayers. (A) Experimental schematic. 225 cm2 2 SOBA fragment organoid cultures in flasks were digested into single cell suspension and then seeded into 96-well BME-coated Transwell chambers at confluence. (B) Brightfield images of SOBA fragment organoid-derived Transwell monolayers 3 days post-seeding. Transwells were established in monolayer growth media or monolayer differentiation media. Scale bar is 100 pm. (C) Trans-epithelial electrical resistance (TEER) of Transwell monolayers cultured in monolayer growth media (solid circles) or monolayer differentiation media (open circles). Data presented are mean ± SD and n = 6 wells.

[0035] FIG. 9 provides images of lung AT2 organoids embedded in hydrogels suspended in the media of BOBA cultures.

[0036] FIG. 10 provides a schematic depicting exemplary conditions for producing hydrogel droplets, which are referred to herein as basement membrane organoid bead assembly (BOBA). Organoids or dissociated organoid cells suspended in a cold liquid hydrogel are dispensed as droplets into room temperature or warmer media, which allows the hydrogel to solidify immediately upon dispensing into the media.

[0037] FIG. 11 provides a schematic depicting exemplary conditions for producing hydrogel filaments, which are referred to herein as syringe-extruded organoid basement membrane assembly (SOBA). Organoids or dissociated organoid cells suspended in a cold liquid hydrogel are extruded as filaments (through a syringe, pipette, or any other dispensing device) into room temperature or warmer media, which allows the hydrogel to solidify immediately upon dispensing into the media. DETAILED DESCRIPTION

[0038] The present disclosure provides tissue-derived epithelial organoids in suspension culture and methods of generating such tissue-derived epithelial organoids. The methods disclosed herein enable the generation of large-scale organoid cultures without the need for tedious manual manipulation, specialized equipment, or automation. By growing organoid cells in hydrogel domes attached to unconventional surfaces, i.e., suspended hydrogels, the volume of hydrogel can be significantly increased, and thus the number of organoid cells that can be grown in a culture vessel can be significantly increased. Furthermore, since the presently disclosed methods do not require deposition of hydrogels on two-dimensional (2D) surfaces, the methods are compatible with a variety of culture vessels (e.g., culture flasks and culture bags), which allows for further culture scale-up, enabling high-throughput.

[0039] The presently disclosed methods allow for the use of suspended hydrogels of various geometries, which can speed up actual culture preparation time. Furthermore, since the organoids are in suspension, they can be sampled, sectioned, or collected at different times during the culture process, which is difficult for conventional organoid cultures that are fixed in a dish. As shown in Example 1, growth of organoids in suspended BME hydrogels is more uniform than in conventionally surface-attached hydrogel domes, where limited molecular diffusion leads to nutrient gradients (Park et al. (2022); Shin et al. (2020)). The suspended hydrogel culture methods disclosed herein generate tissue-derived epithelial organoid models that are more amenable to high-throughput studies, which will be beneficial for both basic science and translational fields.

[0040] For clarity, but without being limiting, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0041] I. Definitions;

[0042] II. Organoids and compositions thereof;

[0043] III. Methods of generating organoids;

[0044] IV. Methods of use;

[0045] V. Systems; and

[0046] VI. Exemplary embodiments

[0047] I. Definitions

[0048] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs. The following references provide one of ordinary skill with a general definition of many of the terms used in this application: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein the following terms have the meanings ascribed to them unless otherwise indicated.

[0049] As used herein, the use of the term “one” or “a” can be taken to mean “one, or more than one”, “at least one”, and “one or more than one”, unless otherwise indicated.

[0050] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per practices in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or within 2-fold.

[0051] The term “antibody” herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0052] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0053] As used herein, the term "culture medium" or "medium" refers to a liquid that covers cells in a culture vessel, such as a culture flask and a multi-well plate, and contains nutrients to nourish the cells. In certain embodiments, the culture medium can also include growth factors or differentiation factors to produce a desired change in the cells.

[0054] The terms "comprise", "comprising", "have", "having", "contain", "containing", "include", "including", and "comprised of" as used herein are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. The disclosure also contemplates other embodiments "comprising", "consisting of, and "consisting essentially of, the embodiments or elements presented herein, whether explicitly set forth or not.

[0055] As used herein, the term "contacting" a cell with a compound (e.g., a therapeutic agent) means exposing the cell to the compound, e.g., placing the compound in a position that allows it to contact the cell. Contacting can be accomplished using any suitable method. For example, but not by way of limitation, "contacting" can be accomplished by adding the compound to a vessel containing the cell. Contacting can also be accomplished by adding the compound to a culture medium that includes the cell. In certain embodiments, "contacting" means exposing a cell (e.g., a cell in a tissue-derived epithelial organoid or in a cell monolayer) to an agent or compound. In certain embodiments, "contacting" means exposing a tissue-derived epithelial organoid or cell monolayer to a potential therapeutic agent or therapeutic agent of interest.

[0056] As used herein, the term "derived from" or "established from" or "differentiated from" when referring to any cell disclosed herein means a cell obtained from a parental cell (e.g., an isolated or purified cell) in a cell line, a tissue (such as a dissociated tissue), or a fluid using any manipulation. Non-limiting examples of such manipulations include single cell isolation, in vitro culture, treatment with, for example, proteins, chemicals, radiation, viral infection, and nucleic acid transfection, and / or mutagenesis. In certain embodiments, the derived cell can be selected from a mixed population by response to a selected course of growth factors, cytokines, adhesion, lack of adhesion, a sorting procedure, or a combination thereof.

[0057] The term "detecting" includes any means of detection, including direct detection and indirect detection.

[0058] As used herein, the term "droplet" when used in reference to a geometric shape refers to a spherical or spheroid shape.

[0059] As used herein, the term "embedded" or "embedding" refers to at least partial covering or surrounding of a cell. In certain embodiments, as used herein, the term "embedded" or "embedding" refers to complete covering or surrounding of a tissue-derived epithelial organoid, for example, with a hydrogel.

[0060] As used herein, the term "expression" or "express" refers to transcription and / or translation of a nucleotide sequence.

[0061] The term "expression vector" is used to denote a linear or circular nucleic acid molecule into which another nucleic acid sequence fragment of suitable size can be integrated. Such nucleic acid fragments can include additional segments that provide for the transcription of a gene encoded by the nucleic acid sequence fragment. Additional segments can include, but are not limited to: promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, origins of replication, and the like, as known in the art. Expression vectors are typically derived from plasmids, cosmids, and viral vectors; the vectors are typically recombinant molecules containing nucleic acid sequences from a variety of sources.

[0062] As used herein, the term "gastrointestinal" refers to oral mucosa, pharynx (larynx), esophagus, stomach, small intestine, large intestine, and rectum.

[0063] As used herein, the term "gastrointestinal stem cell" refers to a stem cell of the gastrointestinal system.

[0064] As used herein, the term "individual" or "subject" refers to a vertebrate or invertebrate animal, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; apes; and monkeys. In certain embodiments, the individual or subject is a human.

[0065] As used herein, the term "intestinal" or "intestine" refers to the rectum, small intestine, and large intestine.

[0066] As used herein, the term "intestinal stem cell" refers to a stem cell of the intestine.

[0067] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur in an artificial environment. An in vitro environment exemplifies, but is not limited to, a cell culture.

[0068] As used herein, the term "in vivo" refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur in a natural environment.

[0069] As used herein, the term "linear" when used in reference to a geometric shape refers to a shape that resembles a line. In certain embodiments, a line can be a straight line, a curved line, or a line of any shape.

[0070] As used herein, the term "isolated" in reference to a cell (e.g., a gastrointestinal stem cell) refers to a cell that has been separated from components of its natural environment.

[0071] As used herein, a "marker" refers to an agent that allows for direct or indirect detection. Markers include, but are not limited to, fluorescent markers, chromogenic markers, electron-dense markers, chemiluminescent markers, and radioactive markers. Non-limiting examples of markers include green fluorescent protein ("GFP"), mCherry, dtTomato, or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12): 905-909 (2005), incorporated herein by reference), 32 P、 14 C、 125 I、 3 H and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase pure enzyme (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, and enzymes that produce a detectable signal, e.g., horseradish peroxidase (HRP), alkaline phosphatase, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD), and heterocyclic oxidases such as uricase and xanthine oxidase.

[0072] The term "nucleic acid" or "polynucleotide" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, e.g., complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (e.g., messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. A nucleic acid molecule can be linear or circular. Moreover, the term nucleic acid molecule includes both the sense and anti-sense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues.

[0073] The term "operably linked", when applied to nucleic acid sequences, e.g., in an expression vector, means that the sequences are arranged so that they function in concert for their intended purpose, i.e., a promoter sequence permits initiation of transcription, which proceeds through the linked coding sequence until terminated by a stop signal.

[0074] As used herein, the term "organoid" refers to a three-dimensional cellular structure obtained by expansion of stem cells (e.g., adult stem cells) that self-organize and can differentiate into functional cell types. See Corro et al. (2020) Am. J. Physiol. Cell Physiol. 319:C151-C165.

[0075] As used herein, the term“plurality” refers to a number greater than one. In certain embodiments, the term“plurality of tissue-derived epithelial stem cells” refers to a number of tissue-derived epithelial stem cells greater than one. For example, but not by way of limitation, a plurality of tissue-derived epithelial stem cells includes at least two tissue-derived epithelial stem cells. In certain non-limiting embodiments, a plurality of tissue-derived epithelial stem cells can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000, or at least about 1,000,000,000 tissue-derived epithelial stem cells.

[0076] As used herein, the term“population of tissue-derived epithelial organoids” refers to a group of at least two tissue-derived epithelial organoids. In certain embodiments, a“population of tissue-derived epithelial organoids” refers to a group of tissue-derived epithelial organoids produced by the same method. In certain non-limiting embodiments, a population of tissue-derived epithelial organoids can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000, or at least about 1,000,000,000 tissue-derived epithelial organoids.

[0077] As used herein, the term“proliferation” refers to an increase in the number of cells.

[0078] As used herein, the term“promoter” denotes a region within a gene with which transcription factors and / or RNA polymerase can bind to control expression of the associated coding sequence. A promoter is typically, but not always, located in the 5' non-coding region of a gene upstream of the translation initiation codon. The promoter region of a gene can include one or more consensus sequences that serve as recognizable binding sites for sequence-specific nucleic acid binding domains of nucleic acid binding proteins. However, such binding sites can also be located in regions outside of the promoter, for example in introns or in enhancer regions downstream of the coding sequence.

[0079] As used herein, the term "solidified" or "solidification" refers to the hardening, thickening, polymerization, and / or increase in rigidity of a substance.

[0080] As used herein, the term "serpentine" when used in reference to a geometric shape refers to a serpentine shape.

[0081] As used herein, the term "spiral" when used in reference to a geometric shape refers to a continuous curve that spirals around a central point or around an axis.

[0082] As used herein, a "subpopulation" refers to a small fraction of a larger quantity of material.

[0083] As used herein, a "tissue-derived epithelial stem cell" refers to an epithelial stem cell obtained from a tissue. In certain embodiments, a tissue-derived epithelial stem cell does not include a pluripotent stem cell (e.g., an induced pluripotent stem cell (iPSC) and an embryonic stem cell (ESC)).

[0084] As used herein, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of the subject matter, beneficial or desired clinical results include, but are not limited to, alleviating or improving one or more signs or symptoms, decreasing the degree of disease, stabilizing (i.e., not worsening) a disease state, preventing a disease, delaying or slowing the progression of a disease, remission of a disease (e.g., cancer), and / or ameliorating or lessening a disease state. A decrease can be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% reduction in the likelihood of a complication, sign, or symptom or progression to another grade. In certain embodiments, "treatment" can also refer to inhibiting the proliferation or progression of a cancer to a more advanced stage by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%.

[0085] II. Organoids and Compositions Thereof

[0086] The present disclosure provides tissue-derived epithelial organoids. In certain embodiments, the tissue-derived epithelial organoids are embedded in a hydrogel that does not adhere to a substrate (e.g., the substrate of a culture vessel). The present disclosure further provides compositions comprising such organoids. In certain embodiments, the tissue-derived epithelial organoids are produced by the methods disclosed herein, e.g., the methods disclosed in Section III.

[0087] In certain embodiments, the present disclosure provides a composition comprising a tissue-derived epithelial organoid and a culture medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel. In certain embodiments, the hydrogel is not attached to a surface of a substrate, e.g., a surface of a cell culture dish and / or a multi-well plate. In certain embodiments, the tissue-derived epithelial organoid embedded within the hydrogel is suspended in the culture medium.

[0088] In certain embodiments, the tissue-derived epithelial organoid is a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a stomach organoid, a kidney organoid, a pancreas organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovary organoid, a taste bud organoid, or a cytotrophoblast organoid. In certain embodiments, the tissue-derived epithelial organoid is a lacrimal gland organoid. In certain embodiments, the tissue-derived epithelial organoid is a tonsil organoid. In certain embodiments, the tissue-derived epithelial organoid is a salivary gland organoid. In certain embodiments, the tissue-derived epithelial organoid is a gastrointestinal organoid. In certain embodiments, the tissue-derived epithelial organoid is a thyroid organoid. In certain embodiments, the tissue-derived epithelial organoid is a lung organoid. In certain embodiments, the tissue-derived epithelial organoid is a mammary gland organoid. In certain embodiments, the tissue-derived epithelial organoid is a liver organoid. In certain embodiments, the tissue-derived epithelial organoid is a bile duct organoid. In certain embodiments, the tissue-derived epithelial organoid is a stomach organoid. In certain embodiments, the tissue-derived epithelial organoid is a kidney organoid. In certain embodiments, the tissue-derived epithelial organoid is a pancreas organoid. In certain embodiments, the tissue-derived epithelial organoid is an endometrial organoid. In certain embodiments, the tissue-derived epithelial organoid is a fallopian tube organoid. In certain embodiments, the tissue-derived epithelial organoid is a cervix organoid. In certain embodiments, the tissue-derived epithelial organoid is a prostate organoid. In certain embodiments, the tissue-derived epithelial organoid is a bladder organoid. In certain embodiments, the tissue-derived epithelial organoid is an ovary organoid. In certain embodiments, the tissue-derived epithelial organoid is a taste bud organoid. In certain embodiments, the tissue-derived epithelial organoid is a cytotrophoblast organoid. In certain embodiments, the tissue-derived epithelial organoid is selected from the group consisting of a lung organoid, a gastrointestinal organoid, a liver organoid, a pancreas organoid, a mammary gland organoid, and combinations thereof.

[0089] In certain embodiments, the tissue-derived epithelial organoid is a lung organoid. In certain embodiments, the tissue-derived epithelial organoid is an alveolar type II (ATII) organoid.

[0090] In certain embodiments, the tissue-derived epithelial organoid is a gastrointestinal organoid. In certain embodiments, the gastrointestinal organoid is an intestinal organoid. For example, but not by way of limitation, the tissue-derived epithelial organoid is a colon or ileum organoid. In certain embodiments, the tissue-derived epithelial organoid is a colon organoid. In certain embodiments, the tissue-derived epithelial organoid is an ileum organoid. In certain embodiments, the tissue-derived epithelial organoid is a rectal organoid. In certain embodiments, the tissue-derived epithelial organoid is an esophageal organoid. In certain embodiments, the tissue-derived epithelial organoid is an oral palatine organoid. In certain embodiments, the gastrointestinal organoid comprises goblet cells and enterocytes.

[0091] In certain embodiments, the tissue-derived epithelial organoid is a breast organoid.

[0092] In certain embodiments, the tissue-derived epithelial organoid is a pancreatic organoid. In certain embodiments, the pancreatic organoid comprises ductal cells, e.g., as disclosed in Example 6.

[0093] In certain embodiments, the tissue-derived epithelial organoid is a liver organoid. In certain embodiments, the liver organoid comprises hepatocytes, e.g., as disclosed in Example 7.

[0094] In certain embodiments, the compositions of the present disclosure can comprise one or more different types of tissue-derived epithelial organoids. For example, but not by way of limitation, the compositions of the present disclosure can comprise colon and ileum organoids. In certain embodiments, the compositions of the present disclosure can comprise liver and bile duct organoids.

[0095] In certain embodiments, a composition of the present disclosure includes about 1 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium, e.g., about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000 or more, about 50,000 or more, about 100,000 or more, about 500,000 or more, about 1,000,000 or more, about 10,000,000 or more, about 100,000,000 or more, or about 1,000,000,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 50 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 100 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 1,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 5,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 10,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 100,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 500,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 1,000,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 10,000,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium. In certain embodiments, a composition of the present disclosure includes about 100,000,000 or more tissue-derived epithelial organoids embedded within a hydrogel suspended in a culture medium.

[0096] In certain embodiments, the hydrogel is a three-dimensional (3D) scaffold. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature greater than about 10 °C. For example, but not by way of limitation, the hydrogel is composed of a material that solidifies at a temperature greater than about 15 °C, greater than about 20 °C, greater than about 25 °C, greater than about 30 °C, greater than about 35 °C, greater than about 40 °C, greater than about 45 °C, or greater than about 50 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature greater than about 25 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature greater than about 30 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature greater than about 35 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature greater than about 40 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature greater than about 45 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature greater than about 50 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature from about 25 °C to about 50 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature from about 25 °C to about 40 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature from about 30 °C to about 50 °C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature from about 30 °C to about 40 °C, for example, about 37 °C.

[0097] In certain embodiments, the hydrogel is a synthetic hydrogel, a natural hydrogel, or a combination thereof. In certain embodiments, the hydrogel is a synthetic hydrogel. In certain embodiments, the hydrogel is a natural hydrogel. In certain embodiments, the hydrogel can be a mixture of a synthetic hydrogel and a natural hydrogel. In certain embodiments, the hydrogel does not include chemically cross-linked proteins and / or polymers.

[0098] In certain embodiments, the hydrogel is a natural hydrogel. In certain embodiments, the natural hydrogel comprises one or more naturally occurring components. For example, but not by way of limitation, the natural hydrogel can comprise one or more proteins, e.g., glycoproteins and / or polysaccharides. Non-limiting examples of glycoproteins include collagen (e.g., type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, and / or type XII collagen), fibronectin, entactin, tenascin, vitronectin, fibulin, hyaluronan, and laminin. In certain embodiments, the natural hydrogel can further comprise one or more components, such as, but not limited to, polysaccharides, water, and / or elastin. In certain embodiments, the natural hydrogel of the present disclosure comprises laminin, entactin, and type IV collagen. In certain embodiments, the natural hydrogel of the present disclosure comprises laminin, entactin, type IV collagen, and heparan sulfate proteoglycans. In certain embodiments, the natural hydrogel comprises extracellular matrix (ECM) secreted and / or derived from epithelial cells, endothelial cells, parietal endoderm-like cells, and / or connective tissue cells.

[0099] In certain embodiments, the hydrogel is a synthetic hydrogel. Non-limiting examples of synthetic hydrogels include synthetic polymers, such as prolamine (Sigma Z378666), polyethylene glycol (PEG), poly(hydroxyethyl methacrylate), poly(ethylene imine), and polyvinyl alcohol (PVA). Additional non-limiting examples of synthetic hydrogels and polymers of such synthetic hydrogels are disclosed in Unal and West (2020) Bioconjugate Chem. 31(10):2253-2271; and Madduma-Bandarage and Madihally (2020) J. of Applied Polymer Science 138(19):e50376, the contents of each of which are incorporated herein by reference in their entirety.

[0100] In certain embodiments, the hydrogel of the present disclosure does not comprise alginate.

[0101] In certain embodiments, the hydrogel can be a commercially available ECM. Non-limiting examples of commercially available ECMs include ECM proteins and Matrigel® basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. In certain embodiments, the ECM is MATRIGEL™ (BD Biosciences), which includes laminin, entactin, and collagen IV. In certain embodiments, the ECM is basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which includes laminin, entactin, collagen IV, and heparan sulfate proteoglycans.

[0102] In certain embodiments, the hydrogel has a protein concentration, e.g., glycoprotein concentration, of greater than about 0.7 mg / ml. In certain embodiments, the hydrogel has a protein concentration, e.g., glycoprotein concentration, of greater than about 1 mg / ml. In certain embodiments, the hydrogel has a protein concentration, e.g., glycoprotein concentration, of greater than about 2 mg / ml. In certain embodiments, the hydrogel has a protein concentration of greater than about 3 mg / ml. In certain embodiments, the hydrogel has a protein concentration of greater than about 4 mg / ml. In certain embodiments, the hydrogel has a protein concentration of greater than about 5 mg / ml. In certain embodiments, the hydrogel has a protein concentration of greater than about 6 mg / ml, greater than about 7 mg / ml, greater than about 8 mg / ml, greater than about 9 mg / ml, or greater than about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of about 0.7 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of about 1 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of about 5 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of about 6 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of no less than 0.7 mg / ml. In certain embodiments, the hydrogel has a protein concentration of no less than 1 mg / ml. In certain embodiments, the hydrogel has a protein concentration of no less than 5 mg / ml. In certain embodiments, the hydrogel has a protein concentration of no less than 6 mg / ml.

[0103] In certain embodiments, the hydrogel comprises greater than about 1 w / v % of the BME component, ECM component, or polymer, on a w / v % basis. For example, and without limitation, the hydrogel comprises greater than about 1 w / v %, greater than about 1.5 w / v %, greater than about 2 w / v %, greater than about 2.5 w / v %, greater than about 3 w / v %, greater than about 3.5 w / v %, greater than about 4 w / v %, greater than about 4.5 w / v %, greater than about 5 w / v %, greater than about 5.5 w / v %, greater than about 6 w / v %, greater than about 6.5 w / v %, greater than about 7 w / v %, greater than about 7.5 w / v %, greater than about 8 w / v %, greater than about 8.5 w / v %, greater than about 9 w / v %, greater than about 9.5 w / v %, greater than about 10 w / v %, greater than about 10.5 w / v %, greater than about 11 w / v %, greater than about 11.5 w / v %, greater than about 12 w / v %, greater than about 12.5 w / v %, greater than about 13 w / v %, greater than about 13.5 w / v %, greater than about 14 w / v %, greater than about 14.5 w / v %, greater than about 15 w / v %, greater than about 15.5 w / v %, greater than about 16 w / v %, greater than about 16.5 w / v %, greater than about 17 w / v %, greater than about 17.5 w / v %, greater than about 18 w / v %, greater than about 18.5 w / v %, greater than about 19 w / v %, greater than about 19.5 w / v %, or greater than about 20 w / v % of the BME component, ECM component, or polymer, on a w / v % basis. In certain embodiments, the hydrogel comprises about 1 w / v % to about 10 w / v % of the BME component, ECM component, or polymer, on a w / v % basis. In certain embodiments, the hydrogel comprises about 2 w / v % to about 10 w / v % of the BME component, ECM component, or polymer, on a w / v % basis. In certain embodiments, the hydrogel comprises about 5 w / v % to about 10 w / v % of the BME component, ECM component, or polymer, on a w / v % basis.

[0104] In certain embodiments, the hydrogel has a storage modulus G' that is equal to or greater than a loss modulus G".

[0105] In certain embodiments, the hydrogel comprising the tissue-derived epithelial organoid and suspended in the culture medium has a geometric shape. In certain embodiments, the geometric shape of the hydrogel has a length, width, and / or diameter greater than about 0.1 mm. In certain embodiments, the geometric shape of the hydrogel has a length greater than about 0.1 mm. In certain embodiments, the geometric shape of the hydrogel has a width greater than about 0.1 mm. In certain embodiments, the geometric shape of the hydrogel has a diameter greater than about 0.1 mm. For example, and without limitation, the geometric shape of the hydrogel has a length, width, and / or diameter greater than about 0.5 mm, greater than about 1 mm, greater than about 1.5 mm, greater than about 2 mm, greater than about 2.5 mm, greater than about 3 mm, greater than about 3.5 mm, greater than about 4 mm, greater than about 4.5 mm, greater than about 5 mm, greater than about 5.5 mm, greater than about 6 mm, greater than about 6.5 mm, greater than about 7.5 mm, greater than about 8 mm, greater than about 8.5 mm, greater than about 9 mm, greater than about 9.5 mm, greater than about 10 mm, greater than about 10.5 mm, greater than about 11 mm, greater than about 11.5 mm, greater than about 12 mm, greater than about 12.5 mm, greater than about 13 mm, greater than about 13.5 mm, greater than about 14 mm, greater than about 14.5 mm, greater than about 15 mm, greater than about 15.5 mm, greater than about 16 mm, greater than about 16.5 mm, greater than about 17.5 mm, greater than about 18 mm, greater than about 18.5 mm, greater than about 19 mm, greater than about 19.5 mm, greater than about 20 mm, greater than about 50 mm, greater than about 100 mm, greater than about 150 mm, greater than about 200 mm, greater than about 250 mm, greater than about 300 mm, greater than about 350 mm, greater than about 400 mm, greater than about 450 mm, greater than about 500 mm, greater than about 550 mm, greater than about 600 mm, greater than about 650 mm, greater than about 700 mm, greater than about 750 mm, greater than about 800 mm, greater than about 850 mm, greater than about 900 mm, greater than about 950 mm, or greater than about 1,000 mm.

[0106] In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 0.1 mm to about 1000 mm, e.g., about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm, or about 1 mm to about 50 mm. In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 0.1 mm to about 20.0 mm. In certain embodiments, the hydrogel has a geometry with a length of about 0.1 mm to about 20.0 mm. In certain embodiments, the hydrogel has a geometry with a width of about 0.1 mm to about 20.0 mm. In certain embodiments, the hydrogel has a geometry with a diameter of about 0.1 mm to about 20.0 mm.For example, and without limitation, the hydrogel has a geometry with a length, width, and / or diameter of about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 0.1 mm to about 1 mm. In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 0.1 mm to about 5 mm. In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 1 mm to about 20 mm. In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 1 mm to about 10 mm.In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 1 mm to about 5 mm. In certain embodiments, the hydrogel has a geometry with a length, width, and / or diameter of about 1 mm to about 4 mm.

[0107] In certain embodiments, the hydrogel suspended in the culture medium is in the shape of a droplet, e.g., as shown in FIG. 6. In certain embodiments, the hydrogel droplet has a diameter of about 0.1 mm to about 20 mm, e.g., about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the hydrogel droplet has a diameter of about 0.1 mm to about 1 mm. In certain embodiments, the hydrogel droplet has a diameter of about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 20 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 10 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 4 mm.In certain embodiments, each hydrogel droplet comprises about 1 or more tissue-derived epithelial organoids, e.g., about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, or about 10,000 or more tissue-derived epithelial organoids.

[0108] In certain embodiments, the hydrogel has a filamentous structure, e.g., as in FIG. 6. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the filamentous structure has a linear shape. In certain embodiments, the filamentous structure has a serpentine shape. In certain embodiments, the filamentous structure has a helical shape. In certain embodiments, the filamentous structure has a length and / or width of about 0.1 mm to about 1000 mm, e.g., about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm, or about 1 mm to about 50 mm.In certain embodiments, the filamentous structure has a length and / or width of about 0.1 mm to about 20 mm, e.g., about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the filamentous structure has a length of about 0.1 mm to about 1 mm. In certain embodiments, the filamentous structure has a length of about 0.1 mm to about 4 mm. In certain embodiments, the filamentous structure has a length of about 1 mm to about 20 mm. In certain embodiments, the filamentous structure has a length of about 1 mm to about 10 mm. In certain embodiments, the filamentous structure has a length of about 1 mm to about 4 mm. In certain embodiments, the filamentous structure has a width of about 0.1 mm to about 1 mm.In certain embodiments, the filamentous structure has a width of about 0.1 mm to about 4 mm. In certain embodiments, the filamentous structure has a width of about 1 mm to about 20 mm. In certain embodiments, the filamentous structure has a width of about 1 mm to about 10 mm. In certain embodiments, the filamentous structure has a width of about 1 mm to about 4 mm.

[0109] In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 20 mm, e.g., about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 20 mm. In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 10 mm. In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 5 mm. In certain embodiments, the filamentous structure has a diameter of about 1 mm to about 20 mm. In certain embodiments, the filamentous structure has a diameter of about 1 mm to about 10 mm. In certain embodiments, the filamentous structure has a diameter of about 1 mm to about 5 mm.

[0110] In certain embodiments, each filamentous structure comprises about 1 or more tissue-derived epithelial organoid, e.g., about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, or about 10,000 or more tissue-derived epithelial organoids.

[0111] In certain embodiments, the composition comprises any suitable cell culture medium. In certain embodiments, the cell culture medium contains components important for supporting the maintenance of cultured cells. In certain embodiments, the cell culture medium for use in the present disclosure can be a nutrient solution that includes standard cell culture ingredients such as, but not limited to, amino acids, vitamins, inorganic salts, carbon energy sources (e.g., glucose), and buffers. In certain embodiments, the culture medium is a differentiation culture medium. In certain embodiments, the culture medium is a growth culture medium. In certain embodiments, the culture medium is a stem cell promoting culture medium. Non-limiting examples of cell culture media are provided in the Examples, e.g., organoid growth medium.

[0112] In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1 : 1 or greater. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1 : 1 to about 1 : 100. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1 : 5 to about 1 : 100, about 1 : 10 to about 1 : 100, about 1 : 15 to about 1 : 100, about 1 : 20 to about 1 : 100, about 1 : 25 to about 1 : 100, about 1 : 30 to about 1 : 100, about 1 : 35 to about 1 : 100, about 1 : 40 to about 1 : 100, about 1 : 45 to about 1 : 100, about 1 : 45 to about 1 : 100, about 1 : 45 to about 1 : 100, about 1 : 45 to about 1 : 100, about 1 : 50 to about 1 : 100, about 1 : 55 to about 1 : 100, about 1 : 60 to about 1 : 100, about 1 : 65 to about 1 : 100, about 1 : 70 to about 1 : 100, about 1 : 75 to about 1 : 100, about 1 : 80 to about 1 : 100, about 1 : 85 to about 1 : 100, about 1 : 90 to about 1 : 100, about 1 : 95 to about 1 : 100, 1 : 5 to about 1 : 50, about 1 : 10 to about 1 : 50, about 1 : 15 to about 1 : 50, about 1 : 20 to about 1 : 50, about 1 : 25 to about 1 : 50, about 1 : 30 to about 1 : 50, about 1 : 35 to about 1 : 50, about 1 : 40 to about 1 : 50, about 1 : 45 to about 1 : 50, about 1 : 1 to about 1 : 95, about 1 : 1 to about 1 : 90, about 1 : 1 to about 1 : 85, about 1 : 1 to about 1 : 80, about 1 : 1 to about 1 : 75, about 1 : 1 to about 1 : 70, about 1 : 1 to about 1 : 65, about 1 : 1 to about 1 : 60, about 1 : 1 to about 1 : 55, about 1 : 1 to about 1 : 50, about 1 : 1 to about 1 : 45, about 1 : 1 to about 1 : 40, about 1 : 1 to about 1 : 35, about 1 : 1 to about 1 : 30, about 1 : 1 to about 1 : 35, about 1 : 1 to about 1 : 30, about 1 : 1 to about 1 : 25, about 1 : 1 to about 1 : 20, about 1 : 1 to about 1 : 15, about 1 : 1 to about 1 : 10, about 1 : 1 to about 1 : 5, about 1 : 5 to about 1 : 75, about 1 : 5 to about 1 : 60, about 1 : 5 to about 1 : 50, about 1 : 1 to about 1 : 40, about 1 : 5 to about 1 : 30, or about 1 : 5 to about 1 : 20.In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:1 to about 1:50. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:2 to about 1:50. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:1 to about 1:20. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:1 to about 1:15. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:1. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:2. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:5. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:10, e.g., as shown in Example 8. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:20. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:30. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:40. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:50. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:60. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:70. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:80. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:90. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) in the composition is about 1:100.

[0113] In certain embodiments, the tissue-derived epithelial organoids are more uniform in size compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded within a hydrogel attached to a substrate). In certain embodiments, the tissue-derived epithelial organoids produced by the methods of the present disclosure are more uniform in size compared to reference tissue-derived epithelial organoids due to differences in nutrient availability as described in Example 1. For example, but not by way of limitation, the tissue-derived epithelial organoids produced by the methods of the present disclosure are more uniform in size compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded within a hydrogel attached to a substrate) in the width of the suspension culture droplet (e.g., the suspended hydrogel droplet). In certain embodiments, the reference tissue-derived epithelial organoids are produced in a hydrogel dome as disclosed in Example 1.

[0114] In certain embodiments, the tissue-derived epithelial organoids (e.g., population of tissue-derived epithelial organoids) of the present disclosure express a marker at a different (e.g., higher or lower) level compared to a reference tissue-derived epithelial organoid (e.g., population of reference tissue-derived epithelial organoids). For example, but not by way of limitation, the tissue-derived epithelial organoids (e.g., population of tissue-derived epithelial organoids) of the present disclosure express a marker at a higher level compared to a reference tissue-derived epithelial organoid (e.g., population of reference tissue-derived epithelial organoids). Alternatively or additionally, in certain embodiments, the tissue-derived epithelial organoids (e.g., population of tissue-derived epithelial organoids) of the present disclosure express a marker at a lower level compared to a reference tissue-derived epithelial organoid (e.g., population of reference tissue-derived epithelial organoids). In certain embodiments, the marker is a stem cell and / or proliferation marker, e.g., a gene associated with stem cells and / or proliferation, such as FIG. 5A. For example, but not by way of limitation, the stem cell and / or proliferation marker is MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, and / or CD44. In certain embodiments, the marker is a differentiation marker, e.g., a gene associated with differentiation, such as FIG. 5B. For example, but not by way of limitation, the differentiation marker is keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and / or smooth muscle actin (SMA). In certain embodiments, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a substrate. For example, but not by way of limitation, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid produced in a hydrogel dome as disclosed in Example 1.

[0115] In certain embodiments, the tissue-derived epithelial organoid is a gastrointestinal organoid, and the differentially expressed marker in the gastrointestinal organoids of the present disclosure is MKI67, LGR5, SOX9, CD44, MUC2, MUC5B, TFF3, KRT20, FABP1, ALPI, and / or CEACAM7.

[0116] In certain embodiments, the tissue-derived epithelial organoid is a mammary organoid, and the differentially expressed markers in the mammary organoids of the present disclosure are EpCAM, CD49f, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and / or smooth muscle actin (SMA).

[0117] In certain embodiments, the tissue-derived epithelial organoid is a pancreatic organoid, and the differentially expressed markers in the pancreatic organoids of the present disclosure are CD133, LGR5, PDX1, SOX9, ALDH1A1, NEUROG3, NKX6.1, keratin 19 (KRT19), MUC1, INS, GCG, and / or AMY.

[0118] In certain embodiments, the tissue-derived epithelial organoid is a liver organoid, and the differentially expressed markers in the liver organoids of the present disclosure are LGR5, ALB, CYP3A4, HNF4A, KRT19, KRT7, and / or SOX9.

[0119] In certain embodiments, a stem cell and / or a proliferation marker is expressed at a higher level by a population of tissue-derived epithelial organoids of the disclosure (e.g., a population of tissue-derived epithelial organoids in a composition of the disclosure) compared to a reference population of tissue-derived epithelial organoids. Non-limiting examples of stem cell and / or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In certain embodiments, the stem cell and / or proliferation marker is selected from the group consisting of MKI67, ASCL2, LGR5, SOX9, SMOC2, CD44, and combinations thereof. In certain embodiments, the stem cell and / or proliferation marker is MKI67. In certain embodiments, the stem cell and / or proliferation marker is ASCL2. In certain embodiments, the stem cell and / or proliferation marker is LGR5. In certain embodiments, the stem cell and / or proliferation marker is SOX9. In certain embodiments, the stem cell and / or proliferation marker is SMOC2. In certain embodiments, the stem cell and / or proliferation marker is CD44. In certain embodiments, the stem cell and / or proliferation marker is EpCAM. In certain embodiments, the stem cell and / or proliferation marker is CD49f. In certain embodiments, the stem cell and / or proliferation marker is CD133. In certain embodiments, the stem cell and / or proliferation marker is ALDH1A1. In certain embodiments, the stem cell and / or proliferation marker is NEUROG3. In certain embodiments, the stem cell and / or proliferation marker is NKX6.1. In certain embodiments, the stem cell and / or proliferation marker is PDX1. In certain embodiments, the stem cell and / or proliferation marker is BMI1. In certain embodiments, the stem cell and / or proliferation marker is expressed at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 110% higher, at least 120% higher, at least 130% higher, at least 140% higher, at least 150% higher, at least 160% higher, at least 170% higher, at least 180% higher, at least 190% higher, at least 200% higher, at least 210% higher, at least 220% higher, at least 230% higher, at least 240% higher, at least 250% higher, at least 260% higher, at least 270% higher, at least 280% higher, at least 290% higher, or at least 300% higher in a population of tissue-derived epithelial organoids of the disclosure compared to the expression level of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids.In certain embodiments, the level of expression of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 50% higher compared to the level of expression of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the level of expression of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 100% higher compared to the level of expression of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the level of expression of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 200% higher compared to the level of expression of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the level of expression of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 300% higher compared to the level of expression of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids.

[0120] In certain embodiments, a differentiation marker is expressed at a lower level by a population of tissue-derived epithelial organoids of the disclosure (e.g., a population of tissue-derived epithelial organoids in a composition of the disclosure) compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of differentiation markers include keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In certain embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, and combinations thereof. In certain embodiments, the differentiation marker is keratin 20 (KRT20). In certain embodiments, the differentiation marker is FABP1. In certain embodiments, the differentiation marker is MUC2. In certain embodiments, the differentiation marker is MUC5B. In certain embodiments, the differentiation marker is TFF3. In certain embodiments, the differentiation marker is ALPI. In certain embodiments, the differentiation marker is SI. In certain embodiments, the differentiation marker is CEACAM7. In certain embodiments, the differentiation marker is keratin 19 (KRT19). In certain embodiments, the differentiation marker is keratin 7 (KRT7). In certain embodiments, the differentiation marker is SOX9. In certain embodiments, the differentiation marker is MUC1. In certain embodiments, the differentiation marker is INS. In certain embodiments, the differentiation marker is GCG. In certain embodiments, the differentiation marker is AMY. In certain embodiments, the differentiation marker is ALB. In certain embodiments, the differentiation marker is CYP3A4. In certain embodiments, the differentiation marker is HNF4A. In certain embodiments, the differentiation marker is cytokeratin 8 (K8). In certain embodiments, the differentiation marker is cytokeratin 18 (K18). In certain embodiments, the differentiation marker is cytokeratin 5 (K5). In certain embodiments, the differentiation marker is cytokeratin 14 (K14). In certain embodiments, the differentiation marker is smooth muscle actin (SMA). In certain embodiments, the differentiation marker is MUC5AC. In certain embodiments, the differentiation marker is MUC6.In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 100% lower, at least 110% lower, at least 120% lower, at least 130% lower, at least 140% lower, at least 150% lower, at least 160% lower, at least 170% lower, at least 180% lower, at least 190% lower, at least 200% lower, at least 210% lower, at least 220% lower, at least 230% lower, at least 240% lower, at least 200% lower, at least 250% lower, at least 260% lower, at least 270% lower, at least 280% lower, at least 290% lower, or at least 300% lower than the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 50% lower than the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 100% lower than the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 200% lower than the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 300% lower than the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids.

[0121] III. Methods of generating organoids

[0122] The present disclosure provides methods of generating tissue-derived epithelial organoids. In certain embodiments, the present disclosure provides methods for generating tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. The present disclosure further provides methods for generating a suspension culture of tissue-derived epithelial organoids. In certain embodiments, the organoids can be generated using the methods described in Example 1 and Examples 4-8, and FIGS. 10 and 11.

[0123] In certain embodiments, the present disclosure provides methods for producing a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a stomach organoid, a kidney organoid, a pancreas organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovary organoid, a taste bud organoid, or a cytothelial layer organoid. In certain embodiments, the present disclosure provides methods for producing a lacrimal gland organoid. In certain embodiments, the present disclosure provides methods for producing a tonsil organoid. In certain embodiments, the present disclosure provides methods for producing a salivary gland organoid. In certain embodiments, the present disclosure provides methods for producing a gastrointestinal organoid. In certain embodiments, the present disclosure provides methods for producing a thyroid organoid. In certain embodiments, the present disclosure provides methods for producing a lung organoid. In certain embodiments, the present disclosure provides methods for producing a mammary gland organoid. In certain embodiments, the present disclosure provides methods for producing a liver organoid. In certain embodiments, the present disclosure provides methods for producing a bile duct organoid. In certain embodiments, the present disclosure provides methods for producing a stomach organoid. In certain embodiments, the present disclosure provides methods for producing a kidney organoid. In certain embodiments, the present disclosure provides methods for producing a pancreas organoid. In certain embodiments, the present disclosure provides methods for producing an endometrial organoid. In certain embodiments, the present disclosure provides methods for producing a fallopian tube organoid. In certain embodiments, the present disclosure provides methods for producing a cervix organoid. In certain embodiments, the present disclosure provides methods for producing a prostate organoid. In certain embodiments, the present disclosure provides methods for producing a bladder organoid. In certain embodiments, the present disclosure provides methods for producing an ovary organoid. In certain embodiments, the present disclosure provides methods for producing a cytothelial layer organoid.

[0124] In certain embodiments, the present disclosure provides methods for producing a tissue-derived epithelial organoid selected from the group consisting of a lung organoid, a gastrointestinal organoid, a liver organoid, a pancreas organoid, a mammary gland organoid, and combinations thereof.

[0125] In certain embodiments, the present disclosure provides methods for generating a gastrointestinal organoid. In certain embodiments, the present disclosure provides methods for generating an intestinal organoid. For example, but not by way of limitation, the intestinal organoid is a colon or ileum organoid. In certain embodiments, the intestinal organoid is a colon organoid. In certain embodiments, the intestinal organoid is an ileum organoid. In certain embodiments, the present disclosure provides methods for generating a rectal organoid. In certain embodiments, the present disclosure provides methods for generating an esophageal organoid. In certain embodiments, the present disclosure provides methods for generating a buccal palatal organoid. In certain embodiments, the methods of the present disclosure can generate two or more different types of tissue-derived epithelial organoids, such as colon and ileum organoids.

[0126] In certain embodiments, the present disclosure provides methods for generating a lung organoid.

[0127] In certain embodiments, the present disclosure provides methods for generating a liver organoid.

[0128] In certain embodiments, the present disclosure provides methods for generating a pancreatic organoid.

[0129] In certain embodiments, the present disclosure provides methods for generating a breast organoid.

[0130] In certain embodiments, the methods for generating a tissue-derived epithelial organoid comprise contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture.

[0131] In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises at least two or more tissue-derived epithelial stem cells. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises at least about 10 or more tissue-derived epithelial stem cells, at least about 100 or more tissue-derived epithelial stem cells, at least about 1,000 or more tissue-derived epithelial stem cells, at least about 10,000 or more tissue-derived epithelial stem cells, at least about 100,000 or more tissue-derived epithelial stem cells, at least about 100,000 or more tissue-derived epithelial stem cells, at least about 1,000,000 or more tissue-derived epithelial stem cells, at least about 10,000,000 or more tissue-derived epithelial stem cells, or at least about 100,000,000 or more tissue-derived epithelial stem cells, or at least about 1,000,000,000 or more tissue-derived epithelial stem cells. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises at least about 10 or more tissue-derived epithelial stem cells per ml of hydrogel, at least about 100 or more tissue-derived epithelial stem cells per ml of hydrogel, at least about 1,000 or more tissue-derived epithelial stem cells per ml of hydrogel, at least about 10,000 or more tissue-derived epithelial stem cells per ml of hydrogel, at least about 100,000 or more tissue-derived epithelial stem cells per ml of hydrogel, at least about 100,000 or more tissue-derived epithelial stem cells per ml of hydrogel, at least about 1,000,000 or more tissue-derived epithelial stem cells per ml of hydrogel, at least about 10,000,000 or more tissue-derived epithelial stem cells per ml of hydrogel, at least about 100,000,000 or more tissue-derived epithelial stem cells per ml of hydrogel, or at least about 1,000,000,000 or more tissue-derived epithelial stem cells per ml of hydrogel. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises at least about 10,000 or more tissue-derived epithelial stem cells per ml of hydrogel.

[0132] In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1 x 10 4 to about 1 x 10 10 tissue-derived epithelial stem cells per ml of hydrogel. For example, and without limitation, the plurality of tissue-derived epithelial stem cells comprises about 1 x 10 4 to about 1 x 10 9 tissue-derived epithelial stem cells per ml of hydrogel, about 1 x 104 to about 1 x 108 about 1 x 10 4 about 1 x 10 7 about 1 x 10 4 about 1 x 10 6 about 1 x 10 4 about 1 x 10 5 about 1 x 10 5 about 1 x 10 10 about 1 x 10 6 about 1 x 10 10 about 1 x 10 7 about 1 x 10 10 about 1 x 10 8 about 1 x 10 10 about 1 x 10 9 about 1 x 10 10 about 1 x 10 5 about 1 x 10 7 about 1 x 10 about 1 x 10 5 about 1 x 10 8 about 1 x 10 4 about 1 x 10 7 about 1 x 10 4 about 1 x 10 6 about 1 x 10

[0133] In certain embodiments, the method can further comprise suspending the hydrogel-tissue derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue derived epithelial stem cell mixture. In certain embodiments, suspending the hydrogel-tissue derived epithelial stem cell mixture in a culture medium comprises submerging a dispensing device containing the hydrogel-tissue derived epithelial stem cell mixture in the culture medium and dispensing the hydrogel-tissue derived epithelial stem cell mixture into the culture medium. In certain embodiments, dispensing the hydrogel-tissue derived epithelial stem cell mixture into the culture medium can be repeated to produce a plurality of and isolated hydrogel-tissue derived epithelial stem cell mixtures suspended in the culture medium. In certain embodiments, the dispensing device can be any device that allows for the delivery of the mixture. In certain embodiments, the delivery device dispenses the mixture in an accurate and controlled manner. Non-limiting examples of dispensing devices include pipettes, droppers, and syringes. In certain embodiments, the dispensing device can be operated manually or automatically. For example, but not by way of limitation, the dispensing device can be operated automatically. In certain embodiments, the dispensing device can be an automated liquid handler. In certain embodiments, the dispensing device can be a liquid handling robot.

[0134] In certain embodiments, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be at least about 1 pl. For example, but not by way of limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be at least about 5 pl, at least about 10 pl, at least about 50 pl, at least about 100 pl, at least about 500 pl, at least about 1 ml, at least about 10 ml, at least about 50 ml, at least about 100 ml, at least about 500 ml, at least about 1 L, at least about 1.5 L, at least about 2 L, at least about 5 L, or at least about 10 L. In certain embodiments, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be from about 1 pl to about 1 L. In certain embodiments, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be from about 1 pl to about 1 ml. For example, but not by way of limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be from about 1 pl to about 900 pl, from about 1 pl to about 800 pl, from about 1 pl to about 700 pl, from about 1 pl to about 600 pl, from about 1 pl to about 500 pl, from about 1 pl to about 400 pl, from about 1 pl to about 300 pl, from about 1 pl to about 200 pl, from about 1 pl to about 100 pl, from about 1 pl to about 10 pl, from about 1 pl to about 900 pl, from about 10 pl to about 1 ml, from about 100 pl to about 1 ml, from about 200 pl to about 1 ml, from about 300 pl to about 1 ml, from about 400 pl to about 1 ml, from about 500 pl to about 1 ml, from about 600 pl to about 1 ml, from about 700 pl to about 1 ml, from about 800 pl to about 1 ml, from about 900 pl to about 1 ml, from about 1 pl to about 50 pl, from about 5 pl to about 20 pl, from about 10 pl to about 100 pl, from about 10 pl to about 500 pl, from about 100 pl to about 200 pl, or from about 100 pl to about 500 pl. In certain embodiments, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be from about 1 pl to about 100 pl.

[0135] In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature greater than about 10 °C. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature greater than about 15 °C, greater than about 20 °C, greater than about 25 °C, greater than about 30 °C, greater than about 35 °C, greater than about 40 °C, greater than about 45 °C, or greater than about 50 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature greater than about 25 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature greater than about 30 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature greater than about 35 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature greater than about 40 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature greater than about 45 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature of about 25 °C to about 50 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature of about 30 °C to about 50 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature of about 25 °C to about 40 °C, e.g., about 37 °C. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature of about 30 °C to about 40 °C, e.g., about 37 °C.

[0136] In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 25°C or less prior to contacting the culture medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 20°C or less prior to contacting the culture medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 15°C or less prior to contacting the culture medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 10°C or less prior to contacting the culture medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 5°C or less prior to contacting the culture medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 25°C, about 24°C, about 23°C, about 22°C, about 21°C, about 20°C, about 19°C, about 18°C, about 17°C, about 16°C, about 15°C, about 14°C, about 13°C, about 12°C, about 11°C, about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C, or about 2°C prior to contacting the culture medium. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture prior to contacting the culture medium is from about 2°C to about 25°C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture prior to contacting the culture medium is from about 2°C to about 20°C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture prior to contacting the culture medium is from about 2°C to about 15°C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture prior to contacting the culture medium is from about 2°C to about 10°C.

[0137] In certain embodiments, the hydrogel can include any solidified material. In certain embodiments, the hydrogel is a synthetic hydrogel, a natural hydrogel, or a combination thereof. In certain embodiments, the hydrogel is a synthetic hydrogel. In certain embodiments, the hydrogel is a natural hydrogel. In certain embodiments, the hydrogel can be a mixture of a synthetic hydrogel and a natural hydrogel. In certain embodiments, the hydrogel does not include chemically cross-linked proteins and / or polymers.

[0138] In certain embodiments, the hydrogel is a natural hydrogel. In certain embodiments, the natural hydrogel comprises one or more naturally occurring components. For example, but not by way of limitation, the natural hydrogel can comprise one or more proteins, e.g., glycoproteins and / or polysaccharides. Non-limiting examples of glycoproteins include collagen (e.g., type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, and / or type XII collagen), fibronectin, entactin, tenascin, vitronectin, fibulin, hyaluronan, and laminin. In certain embodiments, the natural hydrogel can further comprise one or more components, such as, but not limited to, polysaccharides, water, and / or elastin. In certain embodiments, the natural hydrogel of the present disclosure comprises laminin, entactin, and type IV collagen. In certain embodiments, the natural hydrogel of the present disclosure comprises laminin, entactin, type IV collagen, and heparan sulfate proteoglycans. In certain embodiments, the natural hydrogel comprises extracellular matrix (ECM) secreted and / or derived from epithelial cells, endothelial cells, parietal endoderm-like cells, and / or connective tissue cells.

[0139] In certain embodiments, the hydrogel is a synthetic hydrogel. Non-limiting examples of synthetic hydrogels include synthetic polymers, such as prolamine (Sigma Z378666), polyethylene glycol (PEG), poly(hydroxyethyl methacrylate), poly(ethylene imine), and polyvinyl alcohol (PVA). Additional non-limiting examples of synthetic hydrogels and polymers of such synthetic hydrogels are disclosed in Unal and West (2020) Bioconjugate Chem. 31(10):2253-2271; and Madduma-Bandarage and Madihally (2020) J. of Applied Polymer Science 138(19):e50376, the contents of each of which are incorporated herein by reference in their entirety.

[0140] In certain embodiments, the hydrogel of the present disclosure does not comprise alginate.

[0141] In certain embodiments, the hydrogel can be a commercially available ECM. Non-limiting examples of commercially available ECMs include ECM proteins and Matrigel® basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. In certain embodiments, the ECM is MATRIGEL™ (BD Biosciences), which includes laminin, entactin, and collagen IV. In certain embodiments, the ECM is basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which includes laminin, entactin, collagen IV, and heparan sulfate proteoglycans.

[0142] In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration, e.g., glycoprotein concentration, of greater than about 0.7 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration, e.g., glycoprotein concentration, of greater than about 1 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration, e.g., glycoprotein concentration, of greater than about 2 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of greater than about 3 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of greater than about 4 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of greater than about 5 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of greater than about 6 mg / ml, greater than about 7 mg / ml, greater than about 8 mg / ml, greater than about 9 mg / ml, or greater than about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of about 0.7 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of about 1 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of about 5 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of about 6 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of no less than 0.7 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of no less than 1 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of no less than 5 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue derived epithelial stem cell mixture has a protein concentration of no less than 6 mg / ml.

[0143] In certain embodiments, the hydrogel comprises greater than about 1 w / v % of the BME component, ECM component, or polymer, on a w / v % basis. For example, and without limitation, the hydrogel comprises greater than about 1 w / v %, greater than about 1.5 w / v %, greater than about 2 w / v %, greater than about 2.5 w / v %, greater than about 3 w / v %, greater than about 3.5 w / v %, greater than about 4 w / v %, greater than about 4.5 w / v %, greater than about 5 w / v %, greater than about 5.5 w / v %, greater than about 6 w / v %, greater than about 6.5 w / v %, greater than about 7 w / v %, greater than about 7.5 w / v %, greater than about 8 w / v %, greater than about 8.5 w / v %, greater than about 9 w / v %, greater than about 9.5 w / v %, or greater than about 10 w / v %, greater than about 10.5 w / v %, greater than about 11 w / v %, greater than about 11.5 w / v %, greater than about 12 w / v %, greater than about 12.5 w / v %, greater than about 13 w / v %, greater than about 13.5 w / v %, greater than about 14 w / v %, greater than about 14.5 w / v %, greater than about 15 w / v %, greater than about 15.5 w / v %, greater than about 16 w / v %, greater than about 16.5 w / v %, greater than about 17 w / v %, greater than about 17.5 w / v %, greater than about 18 w / v %, greater than about 18.5 w / v %, greater than about 19 w / v %, greater than about 19.5 w / v %, or greater than about 20 w / v % of the BME component, ECM component, or polymer, on a w / v % basis. In certain embodiments, the hydrogel comprises about 1 w / v % to about 10 w / v % of the BME component, ECM component, or polymer, on a w / v % basis. In certain embodiments, the hydrogel comprises about 2 w / v % to about 10 w / v % of the BME component, ECM component, or polymer, on a w / v % basis. In certain embodiments, the hydrogel comprises about 5 w / v % to about 10 w / v % of the BME component, ECM component, or polymer, on a w / v % basis.

[0144] In certain embodiments, the hydrogel has a storage modulus G' that is equal to or greater than a loss modulus G".

[0145] In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) is about 1:1 to about 1:50. In certain embodiments, the ratio of hydrogel volume to media volume (volume ratio) is about 1:2 to about 1:50. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:5 to about 1:50, about 1:10 to about 1:50, about 1:15 to about 1:50, about 1:20 to about 1:50, about 1:25 to about 1:50, about 1:30 to about 1:50, about 1:35 to about 1:50, about 1:40 to about 1:50, about 1:45 to about 1:50, about 1:2 to about 1:45, about 1:2 to about 1:40, about 1:2 to about 1:35, about 1:2 to about 1:30, about 1:2 to about 1:35, about 1:2 to about 1:30, about 1:2 to about 1:25, about 1:2 to about 1:20, about 1:2 to about 1:15, about 1:2 to about 1:10, or about 1:2 to about 1:5. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:2 to about 1:20. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:2 to about 1:15. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:2. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:5. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:10, e.g., as shown in Example 8. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:20. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:30. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:40. In certain embodiments, the ratio of hydrogel volume to media volume is about 1:50.

[0146] In certain embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture is dispensed into the culture medium in a certain geometry. For example, but not by way of limitation, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture includes a length, width, and / or diameter of greater than about 0.1 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture has a length of greater than about 0.1 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture has a width of greater than about 0.1 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture has a diameter of greater than about 0.1 mm. For example, but not by way of limitation, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture has a length, width, and / or diameter of greater than about 0.5 mm, greater than about 1 mm, greater than about 1.5 mm, greater than about 2 mm, greater than about 2.5 mm, greater than about 3 mm, greater than about 3.5 mm, greater than about 4 mm, greater than about 4.5 mm, greater than about 5 mm, greater than about 5.5 mm, greater than about 6 mm, greater than about 6.5 mm, greater than about 7.5 mm, greater than about 8 mm, greater than about 8.5 mm, greater than about 9 mm, greater than about 9.5 mm, greater than about 10 mm, greater than about 10.5 mm, greater than about 11 mm, greater than about 11.5 mm, greater than about 12 mm, greater than about 12.5 mm, greater than about 13 mm, greater than about 13.5 mm, greater than about 14 mm, greater than about 14.5 mm, greater than about 15 mm, greater than about 15.5 mm, greater than about 16 mm, greater than about 16.5 mm, greater than about 17.5 mm, greater than about 18 mm, greater than about 18.5 mm, greater than about 19 mm, greater than about 19.5 mm, greater than about 20 mm, greater than about 50 mm, greater than about 100 mm, greater than about 150 mm, greater than about 200 mm, greater than about 250 mm, greater than about 300 mm, greater than about 350 mm, greater than about 400 mm, greater than about 450 mm, greater than about 500 mm, greater than about 550 mm, greater than about 600 mm, greater than about 650 mm, greater than about 700 mm, greater than about 750 mm, greater than about 800 mm, greater than about 850 mm, greater than about 900 mm, greater than about 950 mm, or greater than about 1,000 mm.

[0147] In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture comprises a length, width, and / or diameter of about 0.1 mm to about 1000 mm, e.g., about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm, or about 1 mm to about 50 mm.In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture comprises a length, width, and / or diameter of about 0.1 mm to about 20 mm, e.g., about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture comprises a length, width, and / or diameter of about 0.1 mm to about 4 mm. In certain embodiments, the geometry of the suspended hydrogel-tissue derived epithelial stem cell mixture comprises a length, width, and / or diameter of about 0.1 mm to about 1 mm. In certain embodiments, the suspended hydrogel-tissue derived epithelial stem cell mixture comprises a length, width, and / or diameter of about 0.1 mm to about 4 mm.In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a length, width, and / or diameter of about 1 mm to about 20 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometry with a length, width, and / or diameter of about 1 mm to about 10 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometry with a length, width, and / or diameter of about 1 mm to about 4 mm.

[0148] In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometry that is spherical or spheroid. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometry that is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the filamentous structure has a linear shape. In certain embodiments, the filamentous structure has a serpentine shape. In certain embodiments, the filamentous structure has a helical shape.

[0149] In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in a culture medium in droplets (see, e.g., FIG. 10). In certain embodiments, the hydrogel droplets have a diameter greater than about 0.1 mm. In certain embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 20 mm, e.g., about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 1 mm. In certain embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel droplets have a diameter of about 1 mm to about 20 mm.In certain embodiments, the hydrogel droplets have a diameter of about 1 mm to about 10 mm. In certain embodiments, the hydrogel droplets have a diameter of about 1 mm to about 4 mm. In certain embodiments, each hydrogel droplet includes about 1 or more tissue-derived epithelial stem cells, for example, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000 or more, about 100,000 or more, about 200,000 or more, about 300,000 or more, about 400,000 or more, about 500,000 or more, about 600,000 or more, about 700,000 or more, about 800,000 or more, about 900,000 or more, about 1,000,000 or more, about 2,000,000 or more, about 3,000,000 or more, about 4,000,000 or more, about 5,000,000 or more, about 6,000,000 or more, about 7,000,000 or more, about 8,000,000 or more, about 9,000,000 or more, about 10,000,000 or more, about 100,000,000 or more, or about 1,000,000,000 or more tissue-derived epithelial stem cells.

[0150] In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has filamentous structures (see, e.g., FIG. 11). In certain embodiments, the filamentous structures have a length and / or width greater than about 0.1 mm. In certain embodiments, the filamentous structures have a length greater than about 0.1 mm. In certain embodiments, the filamentous structures have a width greater than about 0.1 mm. In certain embodiments, the filamentous structures have a length and / or width of about 0.1 mm to about 1000 mm, e.g., about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm, or about 1 mm to about 50 mm.In certain embodiments, the filamentous structure has a length and / or width of about 0.1 mm to about 20 mm, e.g., about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the filamentous structure has a length of about 0.1 mm to about 1 mm. In certain embodiments, the filamentous structure has a length of about 0.1 mm to about 4 mm. In certain embodiments, the filamentous structure has a length of about 1 mm to about 20 mm. In certain embodiments, the filamentous structure has a length of about 1 mm to about 10 mm. In certain embodiments, the filamentous structure has a length of about 1 mm to about 4 mm. In certain embodiments, the filamentous structure has a width of about 0.1 mm to about 1 mm. In certain embodiments, the filamentous structure has a width of about 0.1 mm to about 4 mm.In certain embodiments, the filamentous structure has a width of about 1 mm to about 20 mm. In certain embodiments, the filamentous structure has a width of about 1 mm to about 10 mm. In certain embodiments, the filamentous structure has a width of about 1 mm to about 4 mm.

[0151] In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 20 mm, e.g., about 0.1 mm to about 19 mm, about 0.1 mm to about 18 mm, about 0.1 mm to about 17 mm, about 0.1 mm to about 16 mm, about 0.1 mm to about 15 mm, about 0.1 mm to about 14 mm, about 0.1 mm to about 13 mm, about 0.1 mm to about 12 mm, about 0.1 mm to about 11 mm, about 0.1 mm to about 10 mm, about 0.1 mm to about 9 mm, about 0.1 mm to about 8 mm, about 0.1 mm to about 7 mm, about 0.1 mm to about 6 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 20 mm, about 1 mm to about 20 mm, about 2 mm to about 20 mm, about 3 mm to about 20 mm, about 4 mm to about 20 mm, about 5 mm to about 20 mm, about 6 mm to about 20 mm, about 7 mm to about 20 mm, about 8 mm to about 20 mm, about 9 mm to about 20 mm, about 10 mm to about 20 mm, about 11 mm to about 20 mm, about 12 mm to about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 20 mm. In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 10 mm. In certain embodiments, the filamentous structure has a diameter of about 0.1 mm to about 5 mm. In certain embodiments, the filamentous structure has a diameter of about 1 mm to about 20 mm. In certain embodiments, the filamentous structure has a diameter of about 1 mm to about 10 mm. In certain embodiments, the filamentous structure has a diameter of about 1 mm to about 5 mm, e.g., as disclosed in Example 8.

[0152] In certain embodiments, each filamentous structure comprises about 1 or more tissue-derived epithelial stem cells, e.g., about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000 or more, or about 100,000 or more, about 200,000 or more, about 300,000 or more, about 400,000 or more, about 500,000 or more, about 600,000 or more, about 700,000 or more, about 800,000 or more, about 900,000 or more, about 1,000,000 or more, about 2,000,000 or more, about 3,000,000 or more, about 4,000,000 or more, about 5,000,000 or more, about 6,000,000 or more, about 7,000,000 or more, about 8,000,000 or more, about 9,000,000 or more, about 10,000,000 or more, about 100,000,000 or more, about 1,000,000,000 or more, or about 10,000,000,000 or more tissue-derived epithelial stem cells.

[0153] In certain embodiments, the method can further comprise culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue derived epithelial stem cell mixture in a culture medium to produce a tissue-derived epithelial organoid. In certain embodiments, the cell culture medium contains components important for supporting the maintenance of tissue-derived epithelial stem cells and / or organoids. In certain embodiments, the cell culture medium for use in the present disclosure can be a nutrient solution that includes standard cell culture ingredients such as, but not limited to, amino acids, vitamins, inorganic salts, carbon energy sources (e.g., glucose), and buffers. In certain embodiments, the culture medium is a stem cell promoting medium. In certain embodiments, the culture medium is a cell growth medium. In certain embodiments, the culture medium is a differentiation medium. Culture media known to support the growth of specific tissues and cell types are known in the art and can be used in conjunction with the subject matter disclosed herein. For example, but not by way of limitation, examples of culture media that can be used in the present disclosure are disclosed in Calà et al., Front. Bioeng. Biotechnol. 11: 1058970 (2023) (e.g., Table 1 of Calà et al.), the contents of which are hereby incorporated by reference herein. In examples, such as Example 8, additional non-limiting examples of culture media for use in the present disclosure are provided.

[0154] In certain embodiments, the culture medium is present in a container. Non-limiting examples of containers include a culture dish, a multi-well plate, a conical tube, a reservoir, a culture bag, a bioreactor, or a flask. In certain embodiments, the conical tube is a 50 ml conical tube. In certain embodiments, the multi-well plate is a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate. In certain embodiments, the reservoir is a custom reservoir. In certain embodiments, the flask is a 25 ml flask, a 50 ml flask, a 250 ml flask, or a 600 ml flask. In certain embodiments, the flask is a single flask or a hotel flask. In certain embodiments, the container is composed of a material that minimizes the attachment of the tissue-derived epithelial stem cells and / or hydrogel to the surface of the container. Alternatively or additionally, the container can include a surface coating that minimizes the attachment of the tissue-derived epithelial stem cells and / or hydrogel to the surface of the container.

[0155] In certain embodiments, the method can further comprise fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures comprising tissue derived epithelial organoids. In certain embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture can comprise shearing the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures, for example by pipetting the culture medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture produces structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting filamentous structures produces structures that are shorter in length, width, or both. In certain embodiments, fragmenting filamentous structures produces structures that are at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% shorter in length, width, or both, as compared to the hydrogel-tissue derived epithelial stem cell mixture that was initially extruded, for example filamentous structures.

[0156] The presently disclosed subject matter further provides methods for producing a suspension culture of tissue derived epithelial organoids. In certain embodiments, the method can comprise introducing a mixture comprising a hydrogel and tissue derived epithelial stem cells into a culture medium to produce a suspended mixture. In certain embodiments, the mixture introduced into the culture medium comprises a hydrogel and a plurality of tissue derived epithelial stem cells as described herein. In certain embodiments, the method can further comprise culturing the plurality of tissue derived epithelial stem cells in the mixture in the culture medium to produce tissue derived epithelial organoids in a suspended state as described herein. In certain embodiments, the method can further comprise fragmenting the suspended mixture to produce fragmented structures comprising tissue derived epithelial organoids.

[0157] In certain embodiments, a method for producing a tissue-derived epithelial organoid in suspension culture can include contacting gastrointestinal stem cells (e.g., a plurality of tissue-derived epithelial stem cells) with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture and depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate as droplets. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate to have a filamentous structure. In certain embodiments, the method can further include solidifying the hydrogel-tissue-derived epithelial stem cell mixture to produce a solidified hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the method can include suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the method includes culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce a tissue-derived epithelial organoid. In certain embodiments, the method can further include removing the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate prior to suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the culture medium. In certain embodiments, the method can further include fragmenting the solidified hydrogel-tissue-derived epithelial stem cell mixture to produce a fragmented structure including a tissue-derived epithelial organoid prior to or after suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the culture medium.

[0158] In certain embodiments, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within a tissue fragment, an organoid fragment, or a combination thereof. For example, and without limitation, a tissue fragment and / or an organoid fragment comprising a tissue-derived epithelial stem cell or a plurality of tissue-derived epithelial stem cells can be used to generate a hydrogel-tissue-derived epithelial stem cell mixture. Alternatively or additionally, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells is isolated from a tissue (e.g., a tissue fragment), an organoid fragment (e.g., a tissue-derived epithelial organoid fragment), or a combination thereof. In certain embodiments, the tissue-derived epithelial stem cell does not comprise a pluripotent stem cell (e.g., an induced pluripotent stem cell (iPSC) and an embryonic stem cell (ESC)). In certain embodiments, the tissue-derived epithelial stem cell for use in the present disclosure can be obtained from an in vitro cell culture. In certain embodiments, the tissue fragment can be obtained from a frozen sample or a fresh sample, such as a frozen or fresh tissue sample and / or a frozen or fresh tissue organoid fragment. In certain embodiments, the tissue fragment can be a primary tissue fragment. In certain embodiments, the tissue (e.g., tissue fragment) of an individual can be normal (e.g., non-cancerous and / or non-diseased). In certain embodiments, the tissue (e.g., tissue fragment) of an individual can be abnormal (e.g., cancerous and / or diseased). In certain embodiments, the tissue-derived epithelial stem cell can be isolated from a fragment of a primary tissue.

[0159] In certain embodiments, the primary tissue fragment can be a fragment of lacrimal gland, tonsil, salivary gland, gastrointestinal tissue, thyroid, lung, breast, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, ovary, taste bud, or placenta. In certain embodiments, the tissue-derived epithelial stem cells can be isolated from a tissue (or fragment thereof) selected from the group consisting of lacrimal gland, tonsil, salivary gland, gastrointestinal tissue, thyroid, lung, breast, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, ovary, taste bud, placenta, and combinations thereof. In certain embodiments, the primary tissue fragment can be a fragment of lacrimal gland. In certain embodiments, the primary tissue fragment can be a fragment of tonsil. In certain embodiments, the primary tissue fragment can be a fragment of salivary gland. In certain embodiments, the primary tissue fragment can be a fragment of gastrointestinal tissue. In certain embodiments, the primary tissue fragment can be a fragment of thyroid. In certain embodiments, the primary tissue fragment can be a fragment of lung. In certain embodiments, the primary tissue fragment can be a fragment of breast. In certain embodiments, the primary tissue fragment can be a fragment of liver. In certain embodiments, the primary tissue fragment can be a fragment of bile duct. In certain embodiments, the primary tissue fragment can be a fragment of stomach. In certain embodiments, the primary tissue fragment can be a fragment of kidney. In certain embodiments, the primary tissue fragment can be a fragment of pancreas. In certain embodiments, the primary tissue fragment can be a fragment of endometrium. In certain embodiments, the primary tissue fragment can be a fragment of fallopian tube. In certain embodiments, the primary tissue fragment can be a fragment of cervix. In certain embodiments, the primary tissue fragment can be a fragment of prostate. In certain embodiments, the primary tissue fragment can be a fragment of bladder. In certain embodiments, the primary tissue fragment can be a fragment of ovary. In certain embodiments, the primary tissue fragment can be a taste bud. In certain embodiments, the primary tissue fragment can be a fragment of placenta. In certain embodiments, the primary tissue fragment is selected from the group consisting of a lung tissue fragment, a liver tissue fragment, a gastrointestinal tissue fragment, a breast tissue fragment, a pancreas tissue fragment, and combinations thereof.

[0160] In certain embodiments, the primary tissue fragment can be a fragment of gastrointestinal tissue. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's oral mucosa, pharynx (larynx), esophagus, stomach, small intestine, large intestine, and / or rectum. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's oral mucosa. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's pharynx. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's esophagus. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's stomach. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's rectum. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's small intestine. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's large intestine. In certain embodiments, the tissue fragment can be, for example, a fragment of an individual's colon and / or ileum tissue. For example, but not by way of limitation, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells can be isolated from, for example, an individual's colon and / or ileum tissue. In certain embodiments, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells can be isolated from (e.g., an individual's) colon tissue, for example, to generate a colon organoid. In certain embodiments, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells can be isolated from (e.g., an individual's) ileum tissue, for example, to generate an ileum organoid. In certain embodiments, the tissue of, for example, an individual can be normal (i.e., non-cancerous). For example, but not by way of limitation, the colon and / or ileum tissue of, for example, an individual can be normal (i.e., non-cancerous). In certain embodiments, the tissue of, for example, an individual can be cancerous or diseased. For example, but not by way of limitation, the colon and / or ileum tissue of, for example, an individual can be cancerous and / or diseased colon and / or ileum tissue. In certain embodiments, the esophagus tissue of, for example, an individual can be cancerous and / or diseased esophagus tissue. In certain embodiments, the stomach tissue of, for example, an individual can be cancerous and / or diseased stomach tissue. In certain embodiments, the rectum tissue of, for example, an individual can be cancerous and / or diseased rectum tissue. In certain embodiments, the tissue-derived epithelial organoid can be an individual-derived gastrointestinal tumor organoid.

[0161] In certain embodiments, the primary tissue fragment is a lung tissue fragment.

[0162] In certain embodiments, the primary tissue fragment is a liver tissue fragment.

[0163] In certain embodiments, the primary tissue fragment is a breast tissue fragment.

[0164] In certain embodiments, the primary tissue fragment is a pancreas tissue fragment.

[0165] In certain embodiments, the methods of the present disclosure produce tissue-derived epithelial organoids that are uniform in size compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded within a hydrogel attached to a substrate). In certain embodiments, the tissue-derived epithelial organoids produced by the methods of the present disclosure are more uniform in size compared to reference tissue-derived epithelial organoids due to differences in nutrient availability as described in Example 1. For example, but not by way of limitation, the tissue-derived epithelial organoids produced by the methods of the present disclosure are more uniform in size across the width of a suspension culture droplet (e.g., a suspended hydrogel droplet) compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded within a hydrogel attached to a substrate). In certain embodiments, the reference tissue-derived epithelial organoids are produced in a hydrogel dome as disclosed in Example 1.

[0166] In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different (e.g., higher or lower) level as compared to a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). For example, but not by way of limitation, the tissue-derived epithelial organoids (e.g., a population of tissue-derived epithelial organoids) of the present disclosure express a marker at a higher level as compared to a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). Alternatively or additionally, in certain embodiments, the tissue-derived epithelial organoids (e.g., a population of tissue-derived epithelial organoids) of the present disclosure express a marker at a lower level as compared to a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the marker is a stem cell and / or proliferation marker, e.g., a gene associated with stem cells and / or proliferation. For example, but not by way of limitation, the stem cell and / or proliferation marker is MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, and / or CD44. In certain embodiments, the marker is a differentiation marker, e.g., a gene associated with differentiation. For example, but not by way of limitation, the differentiation marker is keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and / or smooth muscle actin (SMA). In certain embodiments, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a basement. For example, but not by way of limitation, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid produced in a hydrogel dome as disclosed in Example 1.

[0167] In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express stem cell and / or proliferation markers at a higher level compared to a reference population of tissue-derived epithelial organoids. Non-limiting examples of stem cell and / or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, and / or CD44, and combinations thereof. In certain embodiments, the stem cell and / or proliferation marker is selected from the group consisting of MKI67, ASCL2, LGR5, SOX9, SMOC2, CD44, and combinations thereof. In certain embodiments, the stem cell and / or proliferation marker is MKI67. In certain embodiments, the stem cell and / or proliferation marker is ASCL2. In certain embodiments, the stem cell and / or proliferation marker is LGR5. In certain embodiments, the stem cell and / or proliferation marker is SOX9. In certain embodiments, the stem cell and / or proliferation marker is SMOC2. In certain embodiments, the stem cell and / or proliferation marker is CD44. In certain embodiments, the stem cell and / or proliferation marker is EpCAM. In certain embodiments, the stem cell and / or proliferation marker is CD49f. In certain embodiments, the stem cell and / or proliferation marker is CD133. In certain embodiments, the stem cell and / or proliferation marker is ALDH1A1. In certain embodiments, the stem cell and / or proliferation marker is NEUROG3. In certain embodiments, the stem cell and / or proliferation marker is NKX6.1. In certain embodiments, the stem cell and / or proliferation marker is PDX1. In certain embodiments, the stem cell and / or proliferation marker is BMI1. In certain embodiments, the expression level of the stem cell and / or proliferation marker in the population of tissue-derived epithelial organoids produced by the methods of the present disclosure is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 110% higher, at least 120% higher, at least 130% higher, at least 140% higher, at least 150% higher, at least 160% higher, at least 170% higher, at least 180% higher, at least 190% higher, at least 200% higher, at least 210% higher, at least 220% higher, at least 230% higher, at least 240% higher, at least 250% higher, at least 260% higher, at least 270% higher, at least 280% higher, at least 290% higher, or at least 300% higher than the expression level of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids.In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 50% higher expression level of a stem cell and / or proliferation marker compared to the expression level of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 100% higher expression level of a stem cell and / or proliferation marker compared to the expression level of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 200% higher expression level of a stem cell and / or proliferation marker compared to the expression level of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 300% higher expression level of a stem cell and / or proliferation marker compared to the expression level of the stem cell and / or proliferation marker in a reference population of tissue-derived epithelial organoids.

[0168] In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a differentiation marker at a lower level compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of differentiation markers include keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In certain embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, and combinations thereof. In certain embodiments, the differentiation marker is keratin 20 (KRT20). In certain embodiments, the differentiation marker is FABP1. In certain embodiments, the differentiation marker is MUC2. In certain embodiments, the differentiation marker is MUC5B. In certain embodiments, the differentiation marker is TFF3. In certain embodiments, the differentiation marker is ALPI. In certain embodiments, the differentiation marker is SI. In certain embodiments, the differentiation marker is CEACAM7. In certain embodiments, the differentiation marker is keratin 19 (KRT19). In certain embodiments, the differentiation marker is keratin 7 (KRT7). In certain embodiments, the differentiation marker is SOX9. In certain embodiments, the differentiation marker is SOX9. In certain embodiments, the differentiation marker is MUC1. In certain embodiments, the differentiation marker is INS. In certain embodiments, the differentiation marker is GCG. In certain embodiments, the differentiation marker is AMY. In certain embodiments, the differentiation marker is ALB. In certain embodiments, the differentiation marker is CYP3A4. In certain embodiments, the differentiation marker is HNF4A. In certain embodiments, the differentiation marker is cytokeratin 8 (K8). In certain embodiments, the differentiation marker is cytokeratin 18 (K18). In certain embodiments, the differentiation marker is cytokeratin 5 (K5). In certain embodiments, the differentiation marker is cytokeratin 14 (K14). In certain embodiments, the differentiation marker is smooth muscle actin (SMA). In certain embodiments, the differentiation marker is MUC5AC. In certain embodiments, the differentiation marker is MUC6.In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 200%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, or at least 300% lower expression of a differentiation marker compared to the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 50% lower expression of a differentiation marker compared to the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 100% lower expression of a differentiation marker compared to the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 200% lower expression of a differentiation marker compared to the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids. In certain embodiments, the population of tissue-derived epithelial organoids produced by the methods of the present disclosure has at least 300% lower expression of a differentiation marker compared to the expression level of the differentiation marker in a reference population of tissue-derived epithelial organoids.

[0169] In certain embodiments, the method for producing a gastrointestinal organoid comprises contacting a tissue-derived epithelial stem cell with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1 x 10 4 7 ​epithelial stem cells per ml of hydrogel. In certain embodiments, the method can further comprise suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture. For example, and without limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be at least about 10 µL. In certain embodiments, the ratio of the volume of the hydrogel-tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, and without limitation, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of a BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which includes laminin, entactin, collagen IV, and heparan sulfate proteoglycans. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the culture medium in a certain geometric shape. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spheroid. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the culture medium as droplets. In certain embodiments, the method can further comprise culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a culture medium (e.g., colon passaging medium (Intesticult Organoid Growth Medium (OGM, StemCell Technologies Cat. No. 06010) + 10 µM Y27632) or ileum medium (OGM + 10 µM Y27632 + 2.5 µM CHIR99021)) to produce a colon or ileum organoid, respectively. In certain embodiments, the method can further comprise fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce a fragmented structure comprising tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture can comprise shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce the fragmented structure, for example, by pipetting the culture medium containing the suspended hydrogel-tissue-derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture produces structures that are shorter in length and / or width.For example, but not by way of limitation, fragmenting the filamentous structures results in structures that are shorter in length, width, or both. In certain embodiments, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within an organoid fragment. In certain embodiments, the methods of the present disclosure generate a population of tissue-derived epithelial organoids that express a marker at a different (e.g., higher or lower) level as compared to a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the differentially expressed marker is MKI67, LGR5, SOX9, CD44, MUC2, MUC5B, TFF3, KRT20, FABP1, ALPI, and / or CEACAM7.

[0170] In certain embodiments, the method for generating a lung organoid comprises contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1 x 10 4 7 ​epithelial stem cells per ml of hydrogel. In certain embodiments, the method can further comprise suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture. For example, and without limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be at least about 10 µL. In certain embodiments, the ratio of the volume of the hydrogel-tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, and without limitation, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. One non-limiting example of an ECM is MATRIGEL®. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the culture medium in a certain geometric shape. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spheroid-like. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the culture medium as droplets. In certain embodiments, the method can further comprise culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium (e.g., SFFF medium containing 10 µM ROCK inhibitor). In certain embodiments, the method can further comprise fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce a fragmented structure comprising tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture can comprise shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce the fragmented structure, for example, by pipetting the culture medium containing the suspended hydrogel-tissue-derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture produces structures that are shorter in length and / or width. For example, and without limitation, fragmenting a filamentous structure produces structures that are shorter in length, width, or both. In certain embodiments, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within an organoid fragment. In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different (e.g., higher or lower) level as compared to a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids).

[0171] In certain embodiments, a method for generating a mammary organoid comprises contacting tissue-derived epithelial stem cells with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1 x 10 4 tissue-derived epithelial stem cells per ml of hydrogel to about 1 x 10 7about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is comprised of a material that solidifies at a temperature greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of a BME is CULTREX® Low Growth Factor BME Type 2 (Trevigen, 3533-010-02). In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the culture medium in a certain geometric shape. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spheroid-like. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the culture medium as droplets. In certain embodiments, the method can further comprise culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium (e.g., SFFF medium containing 10 µM ROCK inhibitor). In certain embodiments, the method can further comprise fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce a fragmented structure comprising tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture can comprise shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce the fragmented structure, for example, by pipetting the culture medium containing the suspended hydrogel-tissue-derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture produces structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting a filamentous structure produces structures that are shorter in length, width, or both. In certain embodiments, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within an organoid fragment.In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different (e.g., higher or lower) level as compared to a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the differentially expressed marker is EpCAM, CD49f, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and / or smooth muscle actin (SMA).

[0172] In certain embodiments, a method for producing a pancreatic organoid comprises contacting a tissue-derived epithelial stem cell with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 1 x 10 4 7 ​epithelial stem cells per ml of hydrogel. In certain embodiments, the method can further comprise suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture. For example, and without limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be at least about 10 µL. In certain embodiments, the ratio of the volume of the hydrogel-tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, and without limitation, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of a BME is Low Growth Factor BME 2-RGF (Basement Membrane Extract Type 2 3533-010-02; AMSBIO, CULTREX®). In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the culture medium in a certain geometric shape. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spheroid. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the culture medium as droplets. In certain embodiments, the method can further comprise culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in an optimized human pancreatic organoid expansion medium (e.g., comprising 1X N2 and 1X B27 (both from GIBCO), 1.25 mM N-acetyl cysteine (Sigma-Aldrich), 10% RSP01 conditioned serum-free medium, 10 nM human [Leu 15Gastrin I (Sigma-Aldrich), 50 ng / mL EGF (Peprotech), 25 ng / mL Noggin (Peprotech), 100 ng / mL FGF10 (Peprotech), 10 mM Nicotinamide (Sigma-Aldrich), 5 mM A83.01 (Tocris), 10 mM FSK (Tocris), and 3 mM PGE2 (Tocris) and supplemented with 10 mM Rho Kinase Inhibitor (Y27632, Sigma-Aldrich). In certain embodiments, the method can further comprise fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture to produce fragmented structures comprising tissue derived epithelial organoids. In certain embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture can comprise pipetting the suspended hydrogel-tissue derived epithelial stem cell mixture up and down to produce fragmented structures, for example by pipetting the medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture produces structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting filamentous structures produces structures that are shorter in length, width, or both. In certain embodiments, the tissue derived epithelial stem cell or plurality of tissue derived epithelial stem cells used in the present disclosure are contained within an organoid fragment. In certain embodiments, the methods of the present disclosure produce a population of tissue derived epithelial organoids that express a marker at a different (e.g., higher or lower) level as compared to a reference tissue derived epithelial organoid (e.g., a population of reference tissue derived epithelial organoids). In certain embodiments, the tissue derived epithelial organoid is a pancreatic organoid and the differentially expressed marker is CD133, LGR5, PDX1, SOX9, ALDH1A1, NEUROG3, NKX6.1, (keratin 19 (KRT19), MUC1, INS, GCG, and / or AMY.

[0173] In certain embodiments, a method for producing a liver organoid comprises contacting tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises about 3,000 to about 10,000 tissue-derived epithelial stem cells per well in a plate (e.g., a 48-well plate). In certain embodiments, the method can further comprise suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture. For example, and without limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the culture medium can be at least about 10 µL. In certain embodiments, the ratio of the volume of the hydrogel-tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, and without limitation, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. Non-limiting examples of ECMs are MATRIGEL® (BD Biosciences) or Low Growth Factor BME 2 (Basement Membrane Extract, Type 2, Pathclear). In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the culture medium in a certain geometry. In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spheroid. In certain embodiments, the geometry of the suspended hydrogel-tissue-derived epithelial stem cell mixture is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the culture medium as droplets.In certain embodiments, the method can further comprise culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue derived epithelial stem cell mixture in a culture medium, for example comprising AdDMEM / F12 (Invitrogen) supplemented with 1% N2 (GIBCO) and 1% B27 (GIBCO), 1.25 mM N-acetyl cysteine (Sigma), 10 nM Gastrin (Sigma), and growth factors: 50 ng / ml EGF (Peprotech), 10% RSP01 conditioned medium (home-made), 100 ng / ml FGF10 (Peprotech), 25 ng / ml HGF (Peprotech), 10 mM Nicotinamide (Sigma), 5 µM A83.01 (Tocris), and 10 µM FSK (Tocris), supplemented with 25 ng / ml Noggin (Peprotech), 30% Wnt medium (as in Barker et al. Cell Stem Cell 6:25-36 (2010)), and 10 µM (Y27632, Sigma Aldrich) or hES Cell Cloning Recovery Solution (Stemgent) for the first 3 days after isolation to establish the culture. In certain embodiments, the culture medium is subsequently changed to, for example, a medium without Noggin, Wnt, Y27632, and hES Cell Cloning Recovery Solution. In certain embodiments, the liver organoids are seeded and cultured in the above-described medium supplemented with BMP7 (25 ng / ml) for 7 to 10 days. In certain embodiments, the culture medium is subsequently changed to a differentiation medium, for example, comprising AdDMEM / F12 medium supplemented with 1% N2 and 1% B27 and containing EGF (50 ng / ml), Gastrin (10 nM, Sigma), HGF (25 ng / ml), FGF19 (100 ng / ml), A8301 (500 nM), DAPT (10 µM), BMP7 (25 ng / ml), and dexamethasone (30 µM) to generate hepatocyte organoids. In certain embodiments, the method can further comprise fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture to generate fragmented structures comprising tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture can comprise shearing the suspended hydrogel-tissue derived epithelial stem cell mixture to generate the fragmented structures, for example, by pipetting the medium containing the suspended hydrogel-tissue derived epithelial stem cell mixture up and down.In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is fragmented to produce structures that are shorter in length and / or width. For example, but not by way of limitation, filamentous structures are fragmented to produce structures that are shorter in length, width, or both. In certain embodiments, the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells for use in the present disclosure are contained within organoid fragments. In certain embodiments, the methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a marker at a different (e.g., higher or lower) level as compared to a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the tissue-derived epithelial organoids are liver organoids and the differentially expressed marker is LGR5, ALB, CYP3A4, HNF4A, KRT19, KRT7, and / or SOX9.

[0174] In certain embodiments, one or more steps of the disclosed methods can be performed using robotics and / or automated components. In certain embodiments, the methods of the present disclosure can comprise using robotics and / or automated components to produce tissue-derived epithelial organoids. In certain embodiments, one or more steps of the disclosed methods can be performed using robotics and / or automated components to produce tissue-derived epithelial organoids. Non-limiting examples of robotics and / or automated components that can be used in the disclosed methods include an automated liquid handler (e.g., a liquid handling robot), a 3D printer, a syringe pump, an electronic pipette (e.g., with or without a pipetting robot), or a combination thereof. A non-limiting example of an electronic pipette (e.g., with a pipetting robot) is the Assist Plus from Integra Biosciences. In certain embodiments, one or more steps of the disclosed methods can be performed by an automated liquid handler (e.g., a liquid handling robot).

[0175] In certain embodiments, passaging of tissue-derived epithelial organoid cultures produced by the methods of the present disclosure is performed by robotics and / or automated components. In certain embodiments, an automated robot can perform the methods described in “Organoid Maintenance” of Example 1 of the present disclosure (e.g.,), perform any of the following steps: adding TrypLE Express, heating the organoid culture, grinding the organoid culture to dissociate the cells, adding PBS, pelleting the cells, resuspending the cells in BME, cooling the culture, plating the cells, covering the organoids with media, or a combination thereof. In certain embodiments, cell dissociation can be performed using robotics and / or automated components. In certain embodiments, media changes (e.g., during organoid maintenance or during tissue-derived epithelial organoid production) can be performed using robotics and / or automated components.

[0176] In certain embodiments, production of BOBA and / or SOBA in cell culture medium is performed by robotic and / or automated components. In certain embodiments, automated robots can perform the methods described in “suspended hydrogel BOBA culture” and / or “suspended hydrogel SOBA fragment culture” of Example 1 of the disclosure (e.g.,) any of the following steps: heating the culture medium, dispensing organoid cell-BME solution into the warm culture medium as droplets to produce BOBA, dispensing organoid cell-BME solution into the warm culture medium in a linear, serpentine, or spiral motion in the X-Y plane to produce SOBA, grinding the SOBA thread culture to produce SOBA fragments, changing the culture medium, or combinations thereof. In certain embodiments, dispensing organoid cell-BME solution into the warm culture medium to produce BOBA and / or SOBA can be performed using robotic and / or automated components (e.g., can be performed by a liquid handling robot). In certain embodiments, changing the culture medium can be performed using robotic and / or automated components (e.g., can be performed by a liquid handling robot). In certain embodiments, grinding the SOBA thread culture to produce SOBA fragments can be performed using robotic and / or automated components (e.g., can be performed by a liquid handling robot).

[0177] In certain embodiments (e.g., as described herein) high-throughput methods can be performed using robotic and / or automated components. For example, but not by way of limitation, any of the methods of use disclosed herein (e.g., as described in Section IV) can be performed, in part, using robotic and / or automated components. In certain embodiments, methods for screening agents (e.g., therapeutic agents) and methods for performing genomic screening using the disclosed tissue-derived epithelial organoids can be performed, in part, using robotic and / or automated components.

[0178] IV. Methods of use

[0179] The disclosure provides methods of using the disclosed organoids or compositions comprising such organoids. In certain embodiments, the tissue-derived epithelial organoids of the disclosure can be used in screening assays. For example, but not by way of limitation, the disclosure provides methods for screening agents (e.g., therapeutic agents), and methods for performing genomic screening using the disclosed tissue-derived epithelial organoids. In certain embodiments, the tissue-derived epithelial organoids of the disclosure can be used to generate organoid-based models.

[0180] In certain embodiments, the organoids of the present disclosure, or compositions thereof, can be used to identify agents having a therapeutic effect. In certain embodiments, the organoids of the present disclosure, or compositions thereof, can be used to identify therapeutic agents that can be effective in preventing and / or treating a disease. In certain embodiments, the organoids of the present disclosure, or compositions thereof, can be used to identify therapeutic agents that can be effective in ameliorating symptoms of a disease.

[0181] In certain embodiments, the organoids of the present disclosure, or compositions thereof, can be used to study the biology and / or pathogenesis of a disease. For example, and without limitation, the organoids of the present disclosure, or compositions thereof, can be contacted with an agent to study the biology and / or pathogenesis of a disease.

[0182] In certain embodiments, the organoids of the present disclosure, or compositions thereof, can be used to identify agents (e.g., therapeutic agents) that can be toxic. In certain embodiments, the organoids of the present disclosure, or compositions thereof, can be used to identify concentrations of agents (e.g., therapeutic agents) that can be toxic.

[0183] In certain embodiments, the methods for identifying therapeutic agents that can be effective in preventing and / or treating a disease, the methods for identifying therapeutic agents that can be effective in ameliorating symptoms of a disease, and / or the methods for identifying therapeutic agents that can be toxic can comprise contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids with a therapeutic agent. In certain embodiments, the methods can comprise contacting a composition comprising a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids with a therapeutic agent. In certain embodiments, the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids is embedded in a hydrogel suspended in a culture medium, as described herein.

[0184] In certain embodiments, the methods for studying the biology and / or pathogenesis of a disease can comprise contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids with an agent. In certain embodiments, the methods can comprise contacting a composition comprising a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids with an agent. In certain embodiments, the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids is embedded in a hydrogel suspended in a culture medium, as described herein.

[0185] In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 1 minute to about 3 years (e.g., about 15 minutes to about 3 years, about 15 minutes to about 2.5 years, about 15 minutes to about 2 years, about 15 minutes to about 1.5 years, about 15 minutes to about 1 year, about 15 minutes to about 183 days, about 15 minutes to about 150 days, about 15 minutes to about 100 days, about 15 minutes to about 50 days, about 1 day to about 3 years, about 10 days to about 3 years, about 20 days to about 3 years, about 50 days to about 3 years, about 100 days to about 3 years, about 150 days to about 3 years, about 183 days to about 3 years, about 1 year to about 3 years, about 1.5 years to about 3 years, about 2 years to about 3 years, or about 2.5 years to about 3 years). In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 1 minute to about 100 days. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 15 minutes to about 100 days. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 1 minute to about 150 days. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 15 minutes to about 150 days. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 1 minute to about 1 year. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 15 minutes to about 1 year. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 1 minute to about 2 years. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 15 minutes to about 2 years. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 1 minute to about 10 days.In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 15 minutes to about 10 days. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 1 hour to about 10 days, about 12 hours to about 10 days, about 1 day to about 10 days, about 2 days to about 10 days, about 3 days to about 10 days, about 4 days to about 10 days, about 5 days to about 10 days, about 6 days to about 10 days, about 7 days to about 10 days, about 8 days to about 10 days, about 9 days to about 10 days, about 15 minutes to about 10 days, about 15 minutes to about 9 days, about 15 minutes to about 8 days, about 15 minutes to about 7 days, about 15 minutes to about 6 days, about 15 minutes to about 5 days, about 15 minutes to about 4 days, about 15 minutes to about 3 days, about 15 minutes to about 2 days, about 15 minutes to about 1 day, about 1 day to about 5 days, about 1 day to about 2 days, about 2 days to about 5 days, or about 2 days to about 10 days. In certain embodiments, the pharmaceutical agent (e.g., therapeutic agent) is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or compositions thereof) for about 2 days to about 10 days.

[0186] In certain embodiments, the methods can include contacting a population of different tissue-derived epithelial organoids (or compositions thereof) with increasing concentrations of a pharmaceutical agent (e.g., therapeutic agent) to allow for dose response studies.

[0187] In certain embodiments, the pharmaceutical agent can be any pharmaceutical agent of interest. In certain embodiments, the pharmaceutical agent is a molecule known to affect the biology and / or pathogenesis of a disease. Non-limiting examples of pharmaceutical agents that can be used in the disclosed methods include a peptide, a polypeptide, a small molecule, a cell, a gene editing system, or a nucleic acid. In certain embodiments, such pharmaceutical agents can be agents that affect cell signaling, nucleic acid expression, protein expression, cell growth, cell differentiation, and / or cell survival.

[0188] In certain embodiments, the pharmaceutical agent is a therapeutic agent. In certain embodiments, the therapeutic agent can be any therapeutic agent of interest. In certain embodiments, the therapeutic agent is obtained from a library of potential therapeutic agents. Non-limiting examples of therapeutic agents that can be analyzed and / or identified using the disclosed methods include a peptide-based therapeutic agent, a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell-based therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, and combinations thereof.

[0189] In certain embodiments, the therapeutic agent is a peptide-based therapeutic agent. In certain embodiments, the peptide-based therapeutic agent comprises a peptide having a molecular weight of about 5,000 Da or less. Non-limiting examples of peptide-based therapeutic agents include growth factors, anti-infective agents, antifungal agents, antibacterial agents, receptor ligands, and tyrosine kinase inhibitors. Other non-limiting examples of peptide therapeutic agents are disclosed in Wang et al. (2022) Signal Transduction and Targeted Therapy 7:48 (e.g., Tables 1 and 2), the contents of which are incorporated herein by reference in their entirety.

[0190] In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic agent. Non-limiting examples of polypeptide-based therapeutic agents include antibody-based therapeutic agents, such as antibodies and antibody drug conjugates, hormones, and enzymes. In certain embodiments, the antibody can be an agonist antibody or an antagonist antibody. In certain embodiments, the antibody can be an antibody fragment. Non-limiting examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multi-specific antibodies formed from antibody fragments.

[0191] In certain embodiments, the therapeutic agent is a small molecule therapeutic agent. For example, but not by way of limitation, the small molecule therapeutic agent is a compound having a molecular weight of less than about 1,000 Da. In certain embodiments, the small molecule therapeutic agent includes cell cycle modulators, kinase modulators (e.g., kinase inhibitors or activators), enzyme inhibitors, receptor modulators (e.g., receptor inhibitors or activators), anti-infective agents, antifungal agents, antibacterial agents, chemotherapeutic agents, and anti-inflammatory agents.

[0192] In certain embodiments, the therapeutic agent is a cell-based therapeutic agent. Non-limiting examples of cell-based therapeutic agents include bacterial cells and immune cells. In certain embodiments, the immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells (NK cells), and lymphocytes, e.g., B cells and T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and helper T cells). In certain embodiments, the immune cells can be modified immune cells engineered to express a chimeric antigen receptor (CAR) (e.g., CAR T cells and CAR NK cells) or a T cell receptor (TCR) (e.g., heterologous TCR).

[0193] In certain embodiments, the therapeutic agent is a gene regulation system and / or a component of a gene regulation system. In certain embodiments, the gene regulation system and / or component of a gene regulation system is a gene editing system, CRISPRi, a gene expression promoting system, a gene suppression promoting system, a nucleic acid-based therapeutic, a transcription factor, and / or a regulator of post-transcriptional modification.

[0194] In certain embodiments, the therapeutic agent is a gene editing system. Non-limiting examples of gene editing systems include homing endonucleases or meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR gene editing systems. In certain embodiments, the therapeutic agent is a CRISPR gene editing system, such as a CRISPR / Cas9 gene editing system.

[0195] In certain embodiments, the therapeutic agent is a nucleic acid-based therapeutic. Non-limiting examples of nucleic acid-based therapeutics include RNA-based therapeutics, including siRNA, microRNA, RNA aptamer, ribozyme, RNA decoy, and RNAi. In certain embodiments, the nucleic acid-based therapeutic includes DNA-based therapeutics, including antisense oligonucleotides (ASOs) and DNA aptamers.

[0196] In certain embodiments, the method can further comprise analyzing changes in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or cells of the tissue-derived epithelial organoid). In certain embodiments, the method can further comprise analyzing changes in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or cells of the tissue-derived epithelial organoid) that occur in the presence of the agent. For example, and without limitation, the method can further comprise analyzing changes in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids (or cells of the tissue-derived epithelial organoid) that are indicative of the effectiveness and / or toxicity of the therapeutic agent. In certain embodiments, the method comprises analyzing changes in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids that are indicative of the effectiveness and / or toxicity of the therapeutic agent as compared to a population of tissue-derived epithelial organoids or tissue-derived epithelial organoids that are not treated with the therapeutic agent.

[0197] In certain embodiments, the organoids or compositions thereof of the present disclosure can be used to perform genome screening. In certain embodiments, genome screening can be used to identify gene editing systems for generating mutations. Non-limiting examples of mutations include deletions, duplications, insertions, and nucleotide substitutions. In certain embodiments, the method can comprise providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof) and generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid. In certain embodiments, the mutation is generated using a gene regulation system and / or a component of a gene regulation system. In certain embodiments, the gene regulation system and / or component of a gene regulation system is a gene editing system, CRISPRi, RNAi, a gene expression promoting system, a gene suppression promoting system, a nucleic acid-based therapeutic, a transcription factor, and / or a regulator of post-transcriptional modification. In certain embodiments, the gene editing system is a CRISPR system, e.g., a CRISPR / Cas9 gene editing system. In certain embodiments, the method can further comprise analyzing a change in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids associated with the mutation. In certain embodiments, the method comprises analyzing a change in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids associated with the mutation compared to a tissue-derived epithelial organoid or population of tissue-derived epithelial organoids that does not have the mutation.

[0198] In certain embodiments, the present disclosure further provides methods for generating an epithelial cell model using the tissue-derived epithelial organoids of the present disclosure. In certain embodiments, the method can comprise providing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids (or a composition thereof), digesting the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids into single cells, and culturing the single cells in a culture medium to generate a cell monolayer. In certain embodiments, the single cells are cultured on a permeable cell culture insert. In certain embodiments, the culture medium is a differentiation culture medium. In certain embodiments, the culture medium is a stem cell promoting culture medium. In certain embodiments, the culture medium is a cell growth culture medium.

[0199] In certain embodiments, the cell monolayer can be used in any of the methods disclosed herein, e.g., in the screening methods disclosed herein. For example, and without limitation, the cell monolayer can be used to screen for therapeutic agents and for performing genome screening. In certain embodiments, the monolayer of the present disclosure can be used to study the biology and / or pathogenesis of a disease.

[0200] In certain embodiments, a method for screening a therapeutic agent using a cell monolayer of the present disclosure can comprise contacting the cell monolayer and analyzing a change in the cell monolayer that is indicative of effectiveness, disposition, and / or toxicity of the therapeutic agent. In certain embodiments, the method comprises analyzing a change in the cell monolayer compared to a cell monolayer that is not treated with the therapeutic agent that is indicative of effectiveness and / or toxicity of the therapeutic agent.

[0201] In certain embodiments, a method for performing a genomic screen using a cell monolayer of the present disclosure can comprise providing a cell monolayer produced by a method described herein, producing a mutation in the genome of one or more cells of the cell monolayer, and analyzing a change in the cell monolayer associated with the mutation. In certain embodiments, the method comprises analyzing a change in the cell monolayer associated with the mutation compared to a cell monolayer that does not have the mutation.

[0202] In certain embodiments, a change in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) can be a change in cell viability, cell proliferation, organoid size, cell morphology, organoid morphology, invasiveness on a hydrogel, motility, differentiation state, mutational state, karyotype, chromosomal aberration, nucleic acid expression level, protein expression level, nucleic acid modification (e.g., methylation), post-translational modification (e.g., phosphorylation, ubiquitination, and / or glycosylation), activation of a cellular signaling pathway, inhibition of a cellular signaling pathway, enzyme activity (e.g., enzymatic cleavage), chromatin accessibility, histone modification and other epigenetic changes, physical properties of the organoid including permeability of the gastrointestinal epithelial barrier, pH, oxygen tension, and concentrations of other metabolites in the lumen and basement membrane and secreted factors in the hydrogel and / or culture medium, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolic pharmacokinetics and pharmacodynamics, force measurements, measurements of interactions between biomolecules within the organoid, lumen, and culture medium / hydrogel, and membrane potential.

[0203] In certain embodiments, a change in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) can be a change in cell viability.

[0204] In certain embodiments, a change in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) can be a change in cell proliferation.

[0205] In certain embodiments, a change in a tissue-derived epithelial organoid (or cells thereof), a population of tissue-derived epithelial organoids (or cells thereof), or a cell monolayer (or cells thereof) can be a change in organoid size.

[0206] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), the population of tissue-derived epithelial organoids (or cells thereof), or the cell monolayer (or cells thereof) can be a change in a mutation status.

[0207] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), the population of tissue-derived epithelial organoids (or cells thereof), or the cell monolayer (or cells thereof) can be a change in RNA expression level and / or protein expression level.

[0208] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), the population of tissue-derived epithelial organoids (or cells thereof), or the cell monolayer (or cells thereof) can be a change in RNA expression level and / or protein expression level. In certain embodiments, the change in the tissue-derived epithelial organoid, the population of tissue-derived epithelial organoids, or the cell monolayer can be a change in protein expression level of a stem cell and / or proliferation marker. In certain embodiments, the change in the tissue-derived epithelial organoid, the population of tissue-derived epithelial organoids, or the cell monolayer can be a change in RNA expression level of a stem cell and / or proliferation marker. For example, but not limited to, the change can be a change in MKI67 expression, a change in EpCAM expression, a change in CD49f expression, a change in ASCL2 expression, a change in CD133 expression, a change in LGR5 expression, a change in SOX9 expression, a change in ALDH1A1 expression, a change in NEUROG3 expression, a change in NKX6.1 expression, a change in SMOC2 expression, a change in PDX1 expression, a change in BMI1 expression, and / or a change in CD44 expression. In certain embodiments, the change can be a change in MKI67 expression. In certain embodiments, the change can be a change in ASCL2 expression. In certain embodiments, the change can be a change in LGR5 expression. In certain embodiments, the change can be a change in SOX9 expression. In certain embodiments, the change can be a change in SMOC2 expression. In certain embodiments, the change can be a change in CD44 expression. In certain embodiments, the change can be a change in EpCAM expression. In certain embodiments, the change can be a change in CD49f expression. In certain embodiments, the change can be a change in CD133 expression. In certain embodiments, the change can be a change in ALDH1A1 expression. In certain embodiments, the change can be a change in NEUROG3 expression. In certain embodiments, the change can be a change in NKX6.1 expression. In certain embodiments, the change can be a change in PDX1 expression. In certain embodiments, the change can be a change in BMI1 expression.

[0209] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), the population of tissue-derived epithelial organoids (or cells thereof), or the cell monolayer (or cells thereof) can be a change in the RNA expression level and / or the protein expression level of a differentiation marker. In certain embodiments, the change in the tissue-derived epithelial organoid, the population of tissue-derived epithelial organoids, or the cell monolayer can be a change in the RNA expression level of a differentiation marker. In certain embodiments, the change in the tissue-derived epithelial organoid, the population of tissue-derived epithelial organoids, or the cell monolayer can be a change in the protein expression level of a differentiation marker. For example, and without limitation, the change can be a change in keratin 20 (KRT20) expression, a change in FABP1 expression, a change in MUC2 expression, a change in MUC5B expression, a change in MUC5AC expression, a change in MUC6 expression, a change in TFF3 expression, a change in ALPI expression, a change in SI expression, a change in CEACAM7 expression, a change in keratin 19 (KRT19) expression, a change in keratin 7 (KRT7) expression, a change in SOX9 expression, a change in MUC1 expression, a change in INS expression, a change in GCG expression, a change in AMY expression, a change in ALB expression, a change in CYP3A4 expression, a change in HNF4A expression, a change in cytokeratin 8 (K8) expression, a change in cytokeratin 18 (K18) expression, a change in cytokeratin 5 (K5) expression, a change in cytokeratin 14 (K14) expression, and / or a change in smooth muscle actin (SMA) expression. In certain embodiments, the change can be a change in KRT20 expression. In certain embodiments, the change can be a change in FABP1 expression. In certain embodiments, the change can be a change in MUC2 expression. In certain embodiments, the change can be a change in MUC5B expression. In certain embodiments, the change can be a change in TFF3 expression. In certain embodiments, the change can be a change in ALPI expression. In certain embodiments, the change can be a change in SI expression. In certain embodiments, the change can be a change in CEACAM7 expression. In certain embodiments, the change can be a change in keratin 19 (KRT19) expression. In certain embodiments, the change can be a change in keratin 7 (KRT7) expression. In certain embodiments, the change can be a change in SOX9 expression. In certain embodiments, the change can be a change in MUC1 expression. In certain embodiments, the change can be a change in INS expression. In certain embodiments, the change can be a change in GCG expression. In certain embodiments, the change can be a change in AMY expression.In certain embodiments, the change can be a change in ALB expression. In certain embodiments, the change can be a change in CYP3A4 expression. In certain embodiments, the change can be a change in HNF4A expression. In certain embodiments, the change can be a change in cytokeratin 8 (K8) expression. In certain embodiments, the change can be a change in cytokeratin 18 (K18) expression. In certain embodiments, the change can be a change in cytokeratin 5 (K5) expression. In certain embodiments, the change can be a change in cytokeratin 14 (K14) expression. In certain embodiments, the change can be a change in smooth muscle actin (SMA) expression. In certain embodiments, the change can be a change in MUC5AC expression. In certain embodiments, the change can be a change in MUC6 expression.

[0210] In certain embodiments, to determine changes in tissue-derived epithelial organoids, cells of tissue-derived epithelial organoids, and / or monolayers or monolayer cells, tissue-derived epithelial organoid cultures or monolayer cultures can be analyzed by flow cytometry techniques, RNA and protein expression, cytokine and metabolite measurements in organoids and culture media, cell viability and proliferation assays, microscopy to monitor epithelial and immune cell motility, barrier function, migration, proliferation, RNA, protein, and subcellular localization of organelles, cell stiffness, and other assays. Tissue-derived epithelial organoids, cells of tissue-derived epithelial organoids, cell monolayers, and / or cells of cell monolayers can be labeled with dyes or transfected with nucleic acids encoding fluorescent proteins and / or luciferases to enable visualization and quantification of cells using imaging and bioluminescence assays.

[0211] In certain embodiments, the present disclosure provides methods for identifying a therapeutic agent that is effective in treating a disease. In certain embodiments, the methods can comprise (i) contacting (a) a tissue-derived epithelial organoid (or a composition thereof), (b) a population of tissue-derived epithelial organoids (or a composition thereof), (c) a cell monolayer derived from a tissue-derived epithelial organoid, or (d) a population of tissue-derived epithelial organoids with a therapeutic agent, and (ii) analyzing a change in the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids, the change indicating effectiveness and / or toxicity of the therapeutic agent. In certain embodiments, the change indicates effectiveness of the therapeutic agent. In certain embodiments, the change indicates toxicity of the therapeutic agent. For example, but not by way of limitation, if the viability of the tissue-derived epithelial organoid or the cells of the tissue-derived epithelial organoid is reduced in the presence of the therapeutic agent compared to the viability of the tissue-derived epithelial organoid or the cells of the tissue-derived epithelial organoid that is not treated with the therapeutic agent, then the therapeutic agent is indicated to be toxic.

[0212] In certain embodiments, the present disclosure provides methods for performing genome screening. In certain embodiments, the methods can comprise (i) providing (a) a tissue-derived epithelial organoid (or a composition thereof), (b) a population of tissue-derived epithelial organoids (or a composition thereof), or (c) a cell monolayer derived from a tissue-derived epithelial organoid, (ii) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid or cell monolayer, and (iii) analyzing a change associated with the mutation in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids. For example, but not by way of limitation, if a change is observed in the tissue-derived epithelial organoid or cell monolayer comprising one or more cells having a genomic mutation as compared to a reference cell or tissue-derived epithelial organoid, then this indicates that the change is a result of the genomic mutation.

[0213] V. Systems

[0214] The present disclosure further provides systems for use in the present disclosure. In certain embodiments, the present disclosure further provides systems for performing the presently disclosed methods. In certain embodiments, the present disclosure provides systems for producing tissue-derived epithelial organoids, such as lacrimal gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, breast organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreas organoids, endometrial organoids, fallopian tube organoids, cervix organoids, prostate organoids, bladder organoids, ovary organoids, taste bud organoids, or cytotrophoblast organoids. In certain embodiments, the present disclosure provides systems for identifying therapeutic agents that are effective in treating a disease. In certain embodiments, the present disclosure provides systems for performing genome screening. In certain embodiments, the present disclosure provides systems for producing epithelial cell models.

[0215] In certain embodiments, the systems of the present disclosure can comprise robotic and / or automated components capable of performing the presently disclosed methods. In certain embodiments, the systems of the present disclosure comprise robotic and / or automated components capable of producing tissue-derived epithelial organoids. In certain embodiments, the systems of the present disclosure comprise one or more robotic and / or automated components for performing one or more steps of the disclosed methods for producing tissue-derived epithelial organoids. Non-limiting examples of robotic and / or automated components include automated liquid handlers (e.g., liquid handling robots), 3D printers, syringe pumps, or combinations thereof. In certain embodiments, the systems of the present disclosure comprise one or more automated liquid handlers.

[0216] In certain embodiments, robots and / or automated components are used to perform the presently disclosed methods and / or to generate tissue-derived epithelial organoids for high-throughput research. In certain embodiments, a system of the present disclosure can include one or more robots and / or automated components for performing a high-throughput method, e.g., as described herein. For example, and without limitation, a system of the present disclosure can include one or more robots and / or automated components that can be used to perform any of the methods of use disclosed herein. In certain embodiments, a system of the present disclosure can include one or more robots and / or automated components for screening agents (e.g., therapeutic agents) and for performing genomic screening using the disclosed tissue-derived epithelial organoids.

[0217] In certain embodiments, robots and / or automated components are capable of passaging tissue-derived epithelial organoid cultures. In certain embodiments, an automated robot can perform the methods described in “Organoid Maintenance” of Example 1 of the present disclosure (e.g.), perform any of the following steps: adding TrypLE Express, heating the organoid culture, triturating the organoid culture to dissociate the cells, adding PBS, pelleting the cells, resuspending the cells in BME, cooling the culture, plating the cells, covering the organoids with media, or a combination thereof. In certain embodiments, a system of the present disclosure can include one or more robots and / or automated components for dissociating cells. In certain embodiments, a system of the present disclosure can include one or more robots and / or automated components for performing media changes, e.g., during organoid maintenance or during generation of tissue-derived epithelial organoids.

[0218] In certain embodiments, a robot and / or automated component is capable of producing BOBAs and / or SOBAs. In certain embodiments, an automated robot can perform the methods described in “suspended hydrogel BOBA cultures” and / or “suspended hydrogel SOBA fragment cultures” of Example 1 of the disclosure (e.g., perform any of the following steps: heating the culture medium, dispensing organoid cell-BME solution into the warm culture medium as droplets to produce BOBAs, dispensing organoid cell-BME solution into the warm culture medium in a linear, serpentine, or spiral pattern of movement to produce SOBAs, grinding the SOBA thread culture to produce SOBA fragments, medium exchange, or combinations thereof. In certain embodiments, a system of the disclosure can include one or more robots and / or automated components for dispensing organoid cell-BME solution into the warm culture medium to produce BOBAs and / or SOBAs. In certain embodiments, a system of the disclosure can include one or more liquid handling robots for dispensing organoid cell-BME solution into the warm culture medium to produce BOBAs and / or SOBAs. In certain embodiments, a system of the disclosure can include one or more robots and / or automated components for performing medium exchange.

[0219] In certain embodiments, a system of the disclosure can include a tissue-derived epithelial organoid, a population of tissue-derived epithelial organoids, and / or a composition including a tissue-derived epithelial organoid, such as a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a stomach organoid, a kidney organoid, a pancreas organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovary organoid, a taste bud organoid, or a cytotrophoblast organoid. In certain embodiments, the tissue-derived epithelial organoid is selected from the group consisting of a lung organoid, a gastrointestinal organoid, a liver organoid, a pancreas organoid, a mammary gland organoid, and combinations thereof. In certain embodiments, the system includes a tissue-derived epithelial organoid produced from one or more tissue-derived epithelial stem cells. In certain embodiments, the tissue-derived epithelial organoid is provided in a hydrogel. In certain embodiments, the tissue-derived epithelial organoid can be embedded in a hydrogel and suspended in a culture medium. Non-limiting examples of tissue-derived epithelial organoids (or compositions thereof) or methods of producing such tissue-derived epithelial organoids are disclosed in Sections II and III, respectively. In certain embodiments, the tissue-derived epithelial organoid can be provided in a container, such as a culture vessel. In certain embodiments, the tissue-derived epithelial organoid can be provided in a culture vessel, such as a flask and / or a multi-well plate.

[0220] In certain embodiments, the systems of the present disclosure can further comprise instructions for using the tissue-derived epithelial organoid to determine whether a therapeutic agent to be tested is effective in treating a disease. In certain embodiments, the systems of the present disclosure can further comprise instructions for using the tissue-derived epithelial organoid to determine the effect of a genomic mutation. In certain embodiments, the systems of the present disclosure can further comprise instructions for generating an epithelial cell model.

[0221] In certain non-limiting embodiments, the systems of the present disclosure can further comprise one or more reagents and other components (e.g., dyes, antibodies, primers, probes, etc.) to determine a change, such as a change in protein or nucleic acid expression, in the tissue-derived epithelial organoid, in a cell of the tissue-derived epithelial organoid, in a cell monolayer, or in a cell of the cell monolayer. Non-limiting examples of such changes are disclosed in Section III. In certain embodiments, the systems of the present disclosure can comprise one or more robots and / or automated components for analyzing a change in the tissue-derived epithelial organoid, in a cell of the tissue-derived epithelial organoid, in a cell monolayer, or in a cell of the cell monolayer.

[0222] VI. Exemplary Embodiments

[0223] A. The presently disclosed subject matter provides a method of generating a tissue-derived epithelial organoid, the method comprising:

[0224] a) contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture;

[0225] b) suspending the hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture; and

[0226] c) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to generate a tissue-derived epithelial organoid.

[0227] A1. The method of A, wherein a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to generate the hydrogel-tissue-derived epithelial stem cell mixture.

[0228] A2. The method of A1, wherein the plurality of tissue-derived epithelial stem cells comprises about 1 x 10 4 tissue-derived epithelial stem cells per ml of hydrogel. 7 tissue-derived epithelial stem cells per ml of hydrogel.

[0229] A3. The method of A1 or A2, wherein the plurality of tissue-derived epithelial stem cells are comprised within a tissue fragment, an organoid fragment, or a combination thereof.

[0230] A4. The method of any one of A-A2, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells are isolated from a primary epithelial tissue.

[0231] A5. The method of any one of A-A4, wherein the hydrogel solidifies upon contact with the culture medium.

[0232] A6. The method of any one of A-A5, wherein suspending the hydrogel-tissue derived epithelial stem cell mixture in the culture medium comprises submerging a dispensing device containing the hydrogel-tissue derived epithelial stem cell mixture in the culture medium and dispensing the hydrogel-tissue derived epithelial stem cell mixture into the culture medium.

[0233] A7. The method of any one of A-A6, wherein the temperature of the culture medium is about 25 °C to about 50 °C.

[0234] A7-1. The method of any one of A-A7, wherein the temperature of the culture medium is about 30 °C to about 50 °C.

[0235] A8. The method of any one of A-A7-1, wherein the temperature of the hydrogel-tissue derived epithelial stem cell mixture is about 2 °C to about 25 °C.

[0236] A8-1. The method of any one of A-A8, wherein the temperature of the hydrogel-tissue derived epithelial stem cell mixture is about 2 °C to about 20 °C.

[0237] A9. The method of any one of A-A8-1, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a natural hydrogel, and a combination thereof.

[0238] A10. The method of A9, wherein the natural hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

[0239] A11. The method of any one of A-A10, wherein the hydrogel has a storage modulus G’ that is equal to or greater than a loss modulus G’ ’.

[0240] A12. The method of any one of A to A11, wherein the suspended hydrogel-tissue derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter of greater than about 0.1 mm.

[0241] A13. The method of A12, wherein the suspended hydrogel-tissue derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0242] A14. The method of any one of A to A13, wherein the hydrogel-tissue derived epithelial stem cell mixture is suspended in the culture medium as droplets.

[0243] A15. The method of any one of A to A13, wherein the suspended hydrogel-tissue derived epithelial stem cell mixture has a filamentous structure.

[0244] A16. The method of A15, wherein the filamentous structure has a linear, serpentine, or helical shape.

[0245] A17. The method of any one of A to A16, further comprising fragmenting the suspended hydrogel-tissue derived epithelial stem cell mixture to produce a fragmented structure comprising tissue derived epithelial organoids.

[0246] B. A method of producing a suspension culture of tissue derived epithelial organoids, comprising:

[0247] a) introducing a mixture comprising a hydrogel and tissue derived epithelial stem cells into a culture medium to produce a suspended mixture; and

[0248] b) culturing the suspended mixture in the culture medium to produce tissue derived epithelial organoids in a suspended state.

[0249] B1. The method of B, wherein the mixture introduced into the culture medium comprises a hydrogel and a plurality of tissue derived epithelial stem cells.

[0250] B2. The method of B1, wherein the plurality of tissue derived epithelial stem cells comprises about 1 x 10 4 tissue derived epithelial stem cells per ml of hydrogel to about 1 x 10 7 tissue derived epithelial stem cells per ml of hydrogel.

[0251] B3. The method of B1 or B2, wherein the plurality of tissue-derived epithelial stem cells are comprised within a tissue fragment, organoid fragment, or a combination thereof.

[0252] B4. The method of any one of B to B2, wherein the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells are isolated from a primary epithelial tissue.

[0253] B5. The method of any one of B to B4, wherein the hydrogel solidifies upon contact with the culture medium.

[0254] B6. The method of any one of B to B5, wherein introducing the mixture into the culture medium comprises submerging a dispensing device loaded with the mixture in the culture medium and dispensing the mixture into the culture medium.

[0255] B7. The method of any one of B to B6, wherein the temperature of the culture medium is from about 25 °C to about 50 °C.

[0256] B7-1. The method of any one of B to B7, wherein the temperature of the culture medium is from about 30 °C to about 50 °C.

[0257] B8. The method of any one of B to B7-1, wherein the temperature of the mixture is from about 2 °C to about 25 °C.

[0258] B8-1. The method of any one of B to B8, wherein the temperature of the mixture is from about 2 °C to about 20 °C.

[0259] B9. The method of any one of B to B8-1, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a natural hydrogel, and a combination thereof.

[0260] B10. The method of B9, wherein the natural hydrogel comprises a basement membrane extract (BME) or extracellular matrix (ECM) component.

[0261] B11. The method of any one of B to B10, wherein the hydrogel has a storage modulus G’ that is equal to or greater than a loss modulus G’.

[0262] B12. The method of any one of B to B11, wherein the suspended mixture has a geometric shape comprising a length, width, and / or diameter that is greater than about 0.1 mm.

[0263] B13. The method of B12, wherein the suspended mixture has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0264] B14. The method of any one of B to B13, wherein the mixture is introduced into the culture medium in the form of droplets.

[0265] B15. The method of any one of B to B3, wherein the mixture is introduced into the culture medium in the form of a filamentous structure.

[0266] B16. The method of B15, wherein the filamentous structure has a linear, serpentine, or helical shape.

[0267] B17. The method of any one of B to B16, further comprising fragmenting the suspended mixture to produce a fragmented structure comprising tissue-derived epithelial organoids.

[0268] B18. The method of any one of A to B17, wherein the culture medium is present in a vessel selected from the group consisting of a culture dish, a multi-well plate, a conical tube, a receptacle, a culture bag, a bioreactor, or a flask.

[0269] C. A method of producing a suspension culture of tissue-derived epithelial organoids, comprising:

[0270] a) contacting tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture;

[0271] b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate;

[0272] c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to produce a solidified hydrogel-tissue-derived epithelial stem cell mixture;

[0273] d) suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture; and

[0274] e) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce tissue-derived epithelial organoids.

[0275] C1. The method of C, wherein a plurality of tissue-derived epithelial stem cells are contacted with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture.

[0276] C2. The method of C1, wherein the plurality of tissue-derived epithelial stem cells comprises about 1 x 10 4 epithelial stem cells per ml of hydrogel. 7

[0277] C3. The method of C1 or C2, wherein the plurality of tissue-derived epithelial stem cells are comprised within tissue fragments, organoid fragments, or a combination thereof.

[0278] C4. The method of any one of C-C2, wherein the tissue-derived epithelial stem cell or plurality of tissue-derived epithelial stem cells are isolated from a primary epithelial tissue.

[0279] C5. The method of any one of C-C3, further comprising removing the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate prior to suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in culture medium.

[0280] C6. The method of any one of C-C4, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a natural hydrogel, and a combination thereof.

[0281] C7. The method of C6, wherein the natural hydrogel comprises a basement membrane extract (BME) or extracellular matrix (ECM) component.

[0282] C8. The method of any one of C-C7, wherein the hydrogel has a storage modulus G' that is equal to or greater than a loss modulus G".

[0283] C9. The method of any one of C-C8, wherein the solidified hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and / or diameter that is greater than about 0.1 mm.

[0284] C10. The method of C9, wherein the solidified hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and / or diameter that is about 0.1 mm to about 1,000 mm.

[0285] C11. The method of any one of C-C10, wherein the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate as a droplet.

[0286] ​C12. The method of any one of C-C10, wherein the hydrogel-tissue derived epithelial stem cell mixture is deposited onto the substrate to have a filamentous structure.

[0287] C13. The method of C12, wherein the filamentous structure has a linear, serpentine, or helical shape.

[0288] C14. The method of any one of C-C13, further comprising fragmenting the hydrogel-tissue derived epithelial stem cell mixture in culture medium to produce fragmented structures comprising tissue derived epithelial organoids.

[0289] C15. The method of any one of C-C14, wherein the tissue derived epithelial organoids have a uniform morphology compared to reference tissue derived epithelial organoids, wherein the reference tissue derived epithelial organoids are tissue derived epithelial organoids embedded within a hydrogel attached to a substrate.

[0290] C16. The method of C15, wherein the tissue derived epithelial organoids have a uniform size.

[0291] C17. The method of C16, wherein the average diameter of the tissue derived epithelial organoids is more uniform compared to reference tissue derived epithelial organoids.

[0292] C18. The method of any one of A-C17, wherein a stem cell and / or proliferation marker is expressed at a higher level in the population of tissue derived epithelial organoids compared to a population of reference tissue derived epithelial organoids, wherein the reference tissue derived epithelial organoids are tissue derived epithelial organoids embedded within a hydrogel attached to a substrate.

[0293] C19. The method of C18, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof.

[0294] C20. The method of any one of A-C18, wherein a differentiation marker is expressed at a lower level in the population of tissue derived epithelial organoids compared to a population of reference tissue derived epithelial organoids, wherein the reference tissue derived epithelial organoids are tissue derived epithelial organoids embedded within a hydrogel attached to a substrate.

[0295] C21. The method of C20, wherein the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

[0296] D. A tissue-derived epithelial organoid produced by the method of any one of A to C21.

[0297] D1. The tissue-derived epithelial organoid of D, wherein the tissue-derived epithelial organoid is selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a stomach organoid, a kidney organoid, a pancreas organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovary organoid, a taste bud organoid, a cytotrophoblast organoid, and combinations thereof.

[0298] E. A composition comprising a tissue-derived epithelial organoid and a culture medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the culture medium.

[0299] E1. The composition of E, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter of greater than about 0.1 mm.

[0300] E2. The composition of E1, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0301] E3. The composition of any one of E to E2, wherein the hydrogel is a droplet.

[0302] E4. The composition of any one of E to E2, wherein the hydrogel has a filamentous structure.

[0303] E5. The composition of E4, wherein the filamentous structure has a linear, serpentine, or helical shape.

[0304] E6. The composition of any one of E-E5, wherein a stem cell and / or a proliferation marker is expressed at a higher level in the population of tissue-derived epithelial organoids compared to a reference tissue-derived epithelial organoid, wherein the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a substrate.

[0305] E7. The composition of E6, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof.

[0306] E8. The composition of any one of E-E5, wherein a differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids compared to a reference tissue-derived epithelial organoid, wherein the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded within a hydrogel attached to a substrate.

[0307] E9. The composition of E8, wherein the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

[0308] F. A method of screening an agent, comprising:

[0309] a) contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids of D or D1 or a composition of any one of E-E9 with an agent; and

[0310] b) analyzing the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for changes indicative of effectiveness and / or toxicity of the agent.

[0311] F1. The method of F, wherein the agent is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for about 1 minute to about 3 years.

[0312] Fl-1. The method of Fl, wherein the agent is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for about 15 minutes to about 3 years.

[0313] F2. The method of F, Fl, or Fl-1, wherein the agent is a therapeutic agent.

[0314] F3. The method of F2, wherein the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cellular therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof.

[0315] G. A method of performing a genomic screen, comprising:

[0316] a) providing the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids of D or Dl or the composition of any one of E-E9;

[0317] b) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid; and

[0318] c) analyzing a change in the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids associated with the mutation.

[0319] Gl. The method of G, wherein the mutation is generated using a gene regulation system.

[0320] G2. The method of Gl, wherein the gene regulation system is a gene editing system.

[0321] G3. The method of G2, wherein the gene editing system is a CRISPR system.

[0322] G4. The method of any one of F-G2, wherein the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of a cell signaling pathway, inhibition of a cell signaling pathway, enzyme activity, barrier integrity, and combinations thereof.

[0323] H. A method of generating an epithelial cell model, comprising:

[0324] a) providing the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids of D;

[0325] b) digesting the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids into single cells; and

[0326] c) culturing the single cell in a culture medium to produce a cell monolayer.

[0327] H1. The method of H, wherein the single cell is cultured on a permeable cell culture insert.

[0328] H2. The method of H or H1, wherein the culture medium is a differentiation culture medium.

[0329] H3. The method of H or H1, wherein the culture medium is a cell growth or stem cell promoting culture medium.

[0330] I. A method of screening an agent, comprising:

[0331] a) contacting a cell monolayer produced by the method of any one of H-H3 with an agent; and

[0332] b) analyzing a change in the cell monolayer indicative of effectiveness, disposition, and / or toxicity of the agent.

[0333] I1. The method of I, wherein the agent is contacted with the cell monolayer for about 1 minute to about 3 years.

[0334] I1-1. The method of I1, wherein the agent is contacted with the cell monolayer for about 15 minutes to about 3 years.

[0335] I2. The method of I, II, or I1-1, wherein the agent is a therapeutic agent.

[0336] I3. The method of I2, wherein the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cellular therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof.

[0337] J. A method of performing a genomic screen, comprising:

[0338] a) providing a cell monolayer produced by the method of any one of H-H3;

[0339] b) producing a mutation in the genome of one or more cells of the cell monolayer; and

[0340] c) analyzing a change in the cell monolayer associated with the mutation.

[0341] J1. The method of J, wherein the mutation is produced using a gene regulation system.

[0342] J2. The method of J1, wherein the gene regulation system is a gene editing system.

[0343] J3. The method of J2, wherein the gene editing system is a CRISPR system.

[0344] J4. The method of any one of J through J3, wherein the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redox potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of a cell signaling pathway, inhibition of a cell signaling pathway, enzyme activity, barrier integrity, and combinations thereof.

[0345] J1. The method of any one of A through C21 and F through J4 or the composition of any one of E through E9, wherein the tissue fragment is a fragment from a tissue selected from the group consisting of lacrimal gland, tonsil, salivary gland, gastrointestinal tissue, thyroid, lung, breast, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, taste bud, ovary, placenta, and combinations thereof.

[0346] J5. The method of any one of A through C21 and F through J4 or the composition of any one of E through E9, wherein the tissue-derived epithelial stem cell is obtained from a fragment of an organoid selected from the group consisting of lacrimal gland organoid, tonsil organoid, salivary gland organoid, gastrointestinal organoid, thyroid organoid, lung organoid, breast organoid, liver organoid, bile duct organoid, stomach organoid, kidney organoid, pancreas organoid, endometrium organoid, fallopian tube organoid, cervix organoid, prostate organoid, bladder organoid, ovary organoid, taste bud organoid, cytotrophoblast organoid, and combinations thereof.

[0347] K. A system for culturing a tissue-derived epithelial organoid, comprising a tissue-derived epithelial organoid and a culture medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the culture medium.

[0348] K1. The system of K, wherein the tissue-derived epithelial organoid is an intestinal organoid.

[0349] K2. The system of K or K1, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter of greater than about 0.1 mm.

[0350] K3. The system of any one of K through K2, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0351] K4. The system of any one of K to K3, wherein the hydrogel is a droplet.

[0352] K5. The system of any one of K to K3, wherein the hydrogel has a filamentous structure.

[0353] K6. The system of K5, wherein the filamentous structure has a linear, serpentine, or helical shape.

[0354] K7. The system of any one of K to K6, wherein a stem cell and / or a proliferation marker is expressed at a higher level in the population of tissue-derived epithelial organoids compared to a reference population of tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within a hydrogel attached to a substrate.

[0355] K8. The system of K7, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof.

[0356] K9. The system of any one of K to K6, wherein a differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids compared to a reference population of tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within a hydrogel attached to a substrate.

[0357] K10. The system of K9, wherein the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

[0358] K11. The system of any one of K to K10, wherein the tissue-derived epithelial organoid is selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a stomach organoid, a kidney organoid, a pancreas organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovary organoid, a taste bud organoid, a cytotrophoblast organoid, and combinations thereof.

[0359] K12. The system of any one of K to K11, further comprising a robot and / or an automated component.

[0360] K13. The system of K12, wherein the robot and / or the automated component comprises a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof.

[0361] L. The method of any one of A to C21 and F to J5, wherein one or more steps of the method are performed by a robot and / or an automated component.

[0362] L1. The method of L, wherein the robot and / or the automated component comprises a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof.

[0363] L2. The method of L or L1, wherein the robot and / or the automated component is a liquid handling robot.

[0364] M. A method of producing a tissue-derived epithelial organoid of D to D1 or a composition of E to E9, wherein one or more steps of the method are performed by a robot and / or an automated component.

[0365] M1. The method of M, wherein the robot and / or the automated component comprises a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof.

[0366] N. The method of any one of B to B18, wherein introducing the mixture comprising the hydrogel and the tissue-derived epithelial stem cells into the culture medium to produce the suspended mixture is performed by a robot and / or an automated component.

[0367] N1. The method of any one of B to B18, wherein culturing the suspended mixture in the culture medium to produce the tissue-derived epithelial organoid in a suspended state is performed by a robot and / or an automated component.

[0368] N2. The method of N or N1, wherein the robotic and / or automated components comprise a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof.

[0369] N3. The method of N2, wherein the robotic and / or automated components comprise one or more liquid handling robots.

[0370] Example

[0371] The subject matter disclosed herein will be better understood by reference to the following examples, which are offered by way of illustration of the subject matter disclosed herein but not by way of limitation.

[0372] Example 1: Formation of Intestinal Organoids

[0373] This example provides a method for culturing intestinal organoids in a variety of geometrically suspended basement membrane extract (BME) hydrogels. The method simplifies the protocol, increases scalability, enables kinetic sampling, and improves culture uniformity without the need for specialized equipment or additional expertise. The method is compatible with multiple culture formats, and the organoids produced by the method can be used for downstream applications such as performing medium-throughput drug screening and generating Transwell monolayers for barrier assessment, as shown in Example 2 and Example 3. The suspended BME hydrogel culturing method enables a wider range of use and higher throughput of intestinal organoids than was previously possible.

[0374] Methods

[0375] Human intestinal organoid source. Organoid source was as previously described (Pleguezuelos-Manzano et al., (2020)) with some modifications. De-identified human colon and ileum tissue samples from deceased donors were obtained through the Donor Network West. Tissue was washed in Advanced DMEM / F12 media (ThermoFisher), cut into 5 cm x 5 cm sections, and then the epithelium was scraped from the submucosa into culture media and minced with a blade. The solution was pelleted at 450 x g for 5 minutes, then resuspended in Advanced DMEM / F12 media without Mg 2+ or Ca 2+in PBS for 9 minutes (ileum) or 12 minutes (colon) at 37°C with vortexing every 3 to 4 minutes until crypts were released. Crypts were pelleted at 450 x g for 5 minutes, washed in PBS, filtered through sterile gauze, then filtered through a 100 pm cell strainer to remove debris, and pelleted at 450 x g for 5 minutes. Crypts were resuspended in CULTREX® Low Growth Factor Basement Membrane Matrix Type II (BME, R&D Systems Cat# 3533-010-02) on ice, plated in 24-well plates in 50 pL of round bottom, solidified for 15 to 30 minutes at 37°C, then overlaid with 500 pL of colon passaging medium (Intesticult Organoid Growth Medium (OGM, StemCell Technologies Cat# 06010) + 10 pM Y27632) or ileum medium (OGM + 10 pM Y27632 + 2.5 pM CHIR99021). After the first 2 to 3 days of culture, medium was changed every 2 to 3 days, or when the medium turned yellow, with a change to plain OGM for colon cultures and ileum medium for ileum cultures.

[0376] Organoid maintenance. Organoid cultures were passaged every 1 to 2 weeks by digestion with TrypLE Express (ThermoFisher) for 10 minutes at 37°C, then triturated with a P1000 pipette. If necessary, incubation was repeated up to 2 times to obtain a single cell suspension. TrypLE Express was inactivated by dilution with PBS, and cells were pelleted at 450 x g for 3 minutes. Cells were resuspended in BME on ice at 6 x 10 5 cells / mL, plated in 24-well plates in 50 pL of round bottom, and solidified for 15 to 30 minutes at 37°C. The first 2 to 3 days, the round bottom was overlaid with colon passaging medium or ileum medium, and every 2 to 3 days, the medium was changed for colon cultures with plain OGM or for ileum cultures with ileum medium. For colon organoid differentiation, cultures were washed with Advanced DMEM / F12 medium, then overlaid with Intesticult Organoid Differentiation Medium (ODM, StemCell Technologies Cat# 100-0214) + 5 pM DAPT for 5 days, with medium change every 2 to 3 days.

[0377] Suspension hydrogel BOBA cultures. Single organoid cells in BME were prepared on ice as described above. Pre-warmed colon passaging medium or ileum medium was added to 6-well plates (5 mL / well), 100 cm dishes (15 to 30 mL / dish), or 50 mL conical tubes (30 mL / tube) and kept on a warm bead bath at 37 °C. Ultra-low attachment (ULA) or standard tissue culture dishes produced similar results. FIG. 10 provides an exemplary schematic showing the production of BOBAs. To produce suspended BME droplets or BOBAs, an electronic serial pipette (Integra VIAFLO 300) with a wide-bore or cut pipette tip (opening ~2 mm) was used to dispense the organoid cell-BME solution directly into the warm medium at 10 pL volume at slow to medium speed to avoid formation of threads or ribbons instead of droplets. During dispensing, the tip was immediately immersed below the liquid surface and then lifted after each dispensing to ensure droplet separation. For larger format cultures, BOBAs were transferred to flasks by serum pipette or decanting. Medium was changed every 2 to 3 days with plain OGM for colon or ileum medium for ileum. In 6-well plate cultures, a sterile 70 pm cell strainer was placed into the well, the plate was tilted and 4 mL of medium was gently sucked through the strainer. In flask cultures, the flask was tilted at an angle and the BOBAs settled in the corner, then approximately 2 / 3 of the volume of used medium was changed with a serum pipette.

[0378] Suspension hydrogel SOBA and SOBA fragment cultures. Single organoid cells in BME were prepared on ice as described above. Pre-warmed colon passaging medium was added to 6-well plates (5 mL / well), 100 cm dishes (15 to 30 mL / dish) and kept on a warm bead bath. FIG. 11 provides an exemplary schematic showing the production of SOBA threads. To produce suspended SOBA threads, the cell-BME solution was gently sucked into a syringe with a 15 gauge blunt-tipped needle and then directly extruded into the warm medium while moving the immersed needle in a linear, serpentine, or spiral motion in the X-Y plane. The extrusion speed and motion in the X-Y plane can affect the thread length and / or width. To produce SOBA fragments, a 10 mL serum pipette or wide-bore P1000 pipette tip was used to gently grind the SOBA thread culture twice. Additional medium was added to bring the final BME to medium ratio to 1:10. Medium changes were performed as described above for BOBA cultures.

[0379] Brightfield microscopy and image analysis. Cultures were imaged by brightfield microscopy using a THUNDER DMi8 inverted optical microscope (Leica) with 2.5X, 4X, or 10X objectives and a DFC9000 GTC camera (Leica). Images were analyzed using Imaris image analysis software and the Imaris Batch software suite (Oxford Instruments). For automated organoid diameter measurements, the Imaris Surfaces detection module was used with inverted brightfield images. Background and out-of-focus organoids were excluded using a background subtraction step (rolling ball, diameter 19.5 pm). The detected surfaces were then filtered according to four criteria: software assigned quality indicator (>3000 A.U.), minor axis length (>35 pm - to exclude debris), rectangularity (>0.2 - to exclude shadows), and major axis length (between 35 pm and 600 pm to exclude false detections of overlapping organoids). Among the remaining surfaces (at least 40 per analyzed image), the diameter was reported as the longest side of the minimum object oriented bounding box. This process was then performed in batch on all analyzed images. The mean value was calculated for each image, and the mean value of three total experiments with n = 3 replicates was plotted.

[0380] Spatial organoid homogeneity organoid diameter analysis was performed using FIJI (ImageJ). A horizontal rectangular (1.5 mm x 9 mm) ROI was set at the center of each image and divided into 1 mm sections in the X axis. For each section, the diameter spanning the widest point of each organoid was measured manually.

[0381] Immunofluorescence sample preparation and confocal microscopy. 24-well dome cultures were fixed with 2% paraformaldehyde (PFA) in PBS. The domes were detached from the dish using a spatula and transferred to microfuge tubes using a cut P1000 pipette tip. For BOBA cultures, 500 µL of culture was transferred to a microfuge tube using a cut P1000 pipette tip, the media was removed and 2% PFA in PBS was added. The samples were incubated in fixative for 15 to 30 minutes at room temperature and then washed 3 times in PBS. The samples were stained in primary antibody diluted in blocking / permeabilization buffer (3% BSA, 0.1% Triton X-100, 0.02% sodium azide in PBS) for at least 4 hours at room temperature and then washed 3 times in PBS. The primary antibodies used were as follows: a-Ki67 (Invitrogen Cat# MA5-14520), a-MUC2 (Millipore Cat# MABF1989), a-FABP1 (Novus Cat# NBP-87695), and a-CHGA (Novus Cat# NB120-15160). The samples were then incubated with secondary antibody (donkey a-rabbit Alexa Fluor 488 (ThermoFisher Cat# A-21206) or goat a-mouse Alexa Fluor 594 (ThermoFisher Cat# A-11032), DAPI, and Alexa Fluor 660 phalloidin diluted in blocking / permeabilization buffer at room temperature for at least 2 hours. Images were collected on a Stellaris 8 confocal microscope (Leica) using a 40X objective and 3D reconstruction was performed using Imaris image analysis software (Oxford Instruments).

[0382] Transcriptome analysis. For RNA isolation, RNeasy Micro Plus Kit (Qiagen) was used. RLT+ lysis buffer was added to BME domes or pelleted BOBA and stored at -80 °C. RNA isolation was performed using QiaCubeConnect (Qiagen) and RNA was quantified using Nanodrop 8000 (ThermoFisher). Bulk mRNA-seq (NovaSeq PE150) and analysis were performed by Novogene. Reads were aligned using HISAT2 (Mortazavi et al. 2008), differential gene expression analysis was performed using DESeq2 (Anders et al. 2014), and statistical significance was calculated using a negative binomial model and Benjamini-Hochberg FDR correction.

[0383] 96-well plate suspended BME organoid variability. After 9 days of culture, 225 cm 2 Colon organoids SOBA fragments in flasks were gently ground twice using a serum pipette to homogenize the sample without destroying intact organoids. The organoid mixture was transferred to a reagent reservoir and then plated in a 96-well plate in 100 µL / well using a P200 multichannel pipette with wide bore pipette tips. Domes cultures were prepared as described above and plated in 5 µL domes in the center of each well of a 96-well plate using serial pipetting. Cultures were allowed to grow for 7 days before viability measurements were taken. All viability measurements were performed using the Cell Titer Glo 3D Assay kit (Promega) and luminescence was measured on an Ensight plate reader (Perkin Elmer).

[0384] Statistical analysis. All statistical analyses were performed using Prism 9 software (Graphpad) unless otherwise stated. Statistical tests, n, and p values are indicated in the figure legends.

[0385] Results:

[0386] A method for human intestinal organoid culture using suspended BME hydrogels. Conventional intestinal organoid culture methods require deposition of organoid cell solution in cold ECM onto a plastic surface, solidification to form a hydrogel dome in an incubator, and then overlaying with growth media (Figures 1A-1B) (Mahe et al. (2013); Pleguezuelos-Manzano et al. (2020); Sato et al. (2009); Sato et al. (2011)). To address the limitations of scaling this technology, a method was developed in which the cold cell ECM solution is immediately solidified as a floating hydrogel suspended in warm media. A method using suspended BME droplets, BME being Engelbreth-Holm-Swarm (EHS) cell-derived ECM, equivalent to MATRIGEL®, and the method termed BOBA (BME-embedded organoid bead assembly) culture has been shown.

[0387] Intesticult Organoid Growth Media) overlay. Media changes were performed individually for each well once every 2-3 days. For BOBA culture, single cell BME solution was deposited as 10 pL droplets directly into pre-warmed media using an electronic serial pipette with wide bore tip, where the hydrogel immediately solidified into suspended BOBAs. BOBAs can be generated in culture dishes, plates, or conical tubes and easily transferred to larger vessels such as cell culture flasks via serum pipette or decanting (Figures 1A-1B). Media changes were performed for the entire flask by allowing BOBAs to settle, and then replacing the top 75% volume of used media with fresh media.

[0388] Human intestinal organoid cells - BME solution was plated in parallel dome or BOBA cultures and grown in growth media for 9 days. Organoid growth and size were similar in both methods as observed by brightfield microscopy (Figure 1C) and quantified by organoid diameter measurements (Figure 1D). Organoid cell proliferation was also comparable as determined by quantifying cell abundance expressing the proliferation marker Ki-67 (Figures 1E-1F). The BOBA method appeared to enable growth of organoids at a higher density per cm2 than dome culture, as determined by the number of organoids per cm2 (Figure 1G). The BOBA method also appeared to enable growth of organoids at a higher density per mL than dome culture, as determined by the number of organoids per mL (Figure 1H). 2This resulted in greater organoid cell growth in terms of surface area, although statistical significance was observed only in small intestinal (ileum) organoids (Figures 1G to 1H). For colon organoids, dome culture yielded an average of 2.9 x 10⁻⁶ cells. 5 1.5 x 10 live cells / well or 1.5 x 10 in a 24-well plate 5 cells / cm 2 BOBA culture yielded an average of 2.2 x 10⁻⁶. 7 One living cell or at 75cm 2 2.9 x 10 flasks 5 cells / cm 2 (Figure 1G). For ileal organoids, dome culture yielded an average of 4.8 x 10⁻⁶. 5 2.6 x 10 live cells / well or 24-well plate 5 cells / cm 2 BOBA culture yielded an average of 2.9 x 10⁻⁶. 7 One living cell or at 75 cm 2 3.9 x 10 flasks 5 cells / cm 2 ( Figure 1H The number of viable cells per µL of BME hydrogel was similar, indicating that the growth rates of the two methods were comparable at a fixed seeding density (Fig. 1G).

[0389] Organoid differentiation in BOBA culture. A major advantage of intestinal organoid models is their ability to differentiate into various intestinal epithelial cell types by altering the culture medium composition (e.g., by removing stem cell-promoting factors) (Clevers (2016); Schutgens and Clevers (2019); Zachos et al. (2016)). Organoid differentiation in dome and BOBA cultures was compared. Organoids were grown in growth medium for 7 days, then washed and transferred to differentiation medium (Intesticut organoid differentiation medium containing 5 µMDAPT) for 5 days. Bright-field microscopy revealed that, in both culture formats, proliferating organoids in growth medium exhibited a cystic morphology with large lumens (Fig. 2A), while differentiated organoids exhibited a dense spherical morphology with slender columnar cells and small lumens (Fig. 2A).

[0390] Bulk RNA-seq analysis of proliferating and differentiating organoids in dome and BOBA culture. In both culture methods, differentiated organoids downregulated expression of stem cell and proliferation markers (MKI67, LGR5, SOX9, and CD44) and upregulated expression of differentiation markers of goblet cells (MUC2, MUC5B, TFF3) and intestinal epithelial cells (KRT20, FABP1, ALPI, and CEACAM7) relative to proliferating organoids (FIG. 2B). Differentiated cell types were also observed by immunofluorescence (IF) confocal microscopy in both culture formats Figure 2C ).

[0391] Characterization and optimization of BOBA culture conditions. Next, it was evaluated how different culture parameters in the BOBA method would affect organoid growth. Cultures were seeded in 6-well plates (FIGS. 3A-3B) or 25 cm 2 flasks ( Figure 3C ) at various BME volumes and fixed cell seeding densities of 6 x 10 5 cells / mL BME. All BOBAs were generated in 10 pL droplets in 5 mL growth media and evaluated for organoid growth by quantifying organoid diameter and viable cell number after 9 days of culture (FIGS. 3B-3C). In 6-well plates, as the total BME volume per well increased from 0.5 mL to 2 mL, organoid diameter decreased and the ratio of viable cells to BME volume decreased (although not statistically significant). Although the number of cells seeded was higher in the higher BME volume conditions, the total number of viable cells and the ratio of viable cells per cm 2 surface area were comparable across all conditions, indicating less proliferation per seeded cell. Overall, these data suggest that organoid growth is compromised when the BME volume exceeds a threshold of the fixed amount of media in a 6-well plate (FIG. 3B).

[0392] Interestingly, for BOBA cultures in 25 cm 2 flasks, organoids grew equally well in all tested conditions ( Figure 3C ). As the total BME volume per flask increased from 0.5 mL to 2 mL, organoid diameter and the ratio of viable cells to BME volume were similar. The total number of viable cells and the ratio of viable cells per cm 2 surface area increased (although not statistically significant due to inter-experimental variability) as the BME volume in the culture appeared to increase, suggesting that for 6-well plates and 25 cm 2Flasks, the threshold of BME volume to media is different. Both the ratio of BME to media and the type of vessel should be optimized for a specific application.

[0393] Organoid uniformity in BOBA culture. The conventional dome method is known to result in organoid heterogeneity (Pleguezuelos-Manzano et al. (2020); Ringel et al. (2020)). Native ECM hydrogels limit gas and molecular diffusion, resulting in nutrient gradients and heterogeneous organoid growth (Colom et al., (2014) J Biomed Mater Res A 102, 2776-2784; Park et al., (2022); Shin et al. (2020)). It was observed that organoids grew more uniformly in BOBA culture than in Dome culture (Fig. 4). Colon organoid cultures were imaged by brightfield microscopy at the deepest point of each format - the bottom Z-plane of the dome or the center Z-plane of the BOBA - and the average organoid diameter across a rectangular ROI was quantified for each hydrogel center (Fig. 4C). Consistent with previous reports (Park et al. (2022); Shin et al. (2020)), Dome cultured organoids were larger at the edge of the hydrogel and smaller at the core (Fig. 4B-4E). However, organoids in BOBA grew comparable sizes across the hydrogel (Fig. 4B-4E).

[0394] It was also observed that organoids in the core of dome cultures often had a compact spherical acellular morphology, which is often indicative of differentiation (Fig. 4B). Bulk RNA-seq analysis indicated that dome cultured organoids had lower expression of stem cell and proliferation markers and higher expression of intestinal epithelial cell markers relative to their BOBA cultured counterparts, supporting the hypothesis that a subpopulation of differentiated organoids can exist in dome cultures (Fig. 5).

[0395] Alternative geometries of suspended BME hydrogels. While the BOBA method has significant scale-up advantages over the conventional dome method, large-scale culture expansion of suspended BOBA hydrogel droplets remains a labor-intensive effort without an automated liquid handler. To reduce the time and labor required for suspended BME hydrogel culture, alternative hydrogel geometries were designed, specifically hydrogel filaments. Extruded filaments have been used in the field of bioprinting to spatially control cell growth or build 3D hydrogel structures in a layer-by-layer assembly, but often rely on attachment to a surface (Kolesky et al. (2014) Adv. Mater. 26, 2966-2966; Kolesky et al. (2016) Proc National Acad Sci 113, 3179-3184). To produce hydrogel filaments containing organoid cells, called SOBA (syringe extruded organoid BME assembly), a cold cell-BME solution was loaded into a syringe with a 15 gauge (1.37 mm inner diameter) blunt tip and then injected directly into a warm culture medium while moving the tip across the X-Y plane (e.g., in a linear, serpentine, or spiral pattern). By gently grinding the SOBA culture with a wide-bore P1000 tip or a 10 mL serological pipette, filament fragments that more closely approximate the BOBA geometry were also produced, called SOBA fragments.

[0396] Organoids grown in BOBA, SOBA, or SOBA fragment cultures for 9 days showed similar growth as measured by brightfield microscopy (FIG. 6A), organoid diameter measurements (FIGS. 6B-6C), and live cell counts (FIG. 6D). Figure 6D SOBA and SOBA fragment formats produced comparable organoid growth relative to BOBA droplets while enabling faster and less laborious culture preparation. SOBA fragments were more evenly dispersed in the culture medium compared to SOBA cultures, making single cultures easier to split, sample, or aliquot.

[0397] Discussion:

[0398] This example describes the development of a suspended BME hydrogel culture method for human intestinal epithelial organoids that overcomes several challenges associated with conventional surface-attached dome hydrogel culture methods. The BOBA, SOBA, and SOBA filament methods simplify and speed up the protocol compared to dome methods, enable compatibility with scalable culture vessels, allow kinetic culture sampling, and improve organoid culture uniformity.

[0399] Several suspension culture protocols have been proposed for intestinal organoids and tumoroid scale-up, but in these methods, organoids are cultured in liquid media containing low concentrations of dissolved MATRIGEL® that do not form a complete hydrogel (Hirokawa et al. (2021) Commun Biology 4, 1067; Price et al. (2022) Sci Rep-Uk 12, 5571). This is in contrast to the presently disclosed method, which relies on the formation of a fully solidified, insoluble hydrogel. Furthermore, previous studies have shown that while organoids and tumoroids can grow in 5% soluble MATRIGEL® solution, concentrations higher than 5% result in reduced growth. Moreover, intestinal organoids grown in 5% MATRIGEL® exhibit reversed epithelial polarity (Hirokawa et al. (2021)), which is consistent with previous reports of organoid polarity reversal under low ECM conditions (Co et al. (2019) Cell Reports 26, 2509-2520.e4).

[0400] The BOBA method offers several advantages over conventional dome methods. First, it simplifies the protocol. Since the hydrogel solidifies directly in the culture medium, there is no need for a separate solidification incubation step, saving time and labor. Second, it reduces the amount of surface area of the vessel and the technical precision required for plating domes. By reducing the dependence on available surface area, the BOBA method takes advantage of all three dimensions of the culture vessel, increasing the hydrogel volume and organoid cells per culture. The BOBA method facilitates large-scale culture because it enables compatibility with flasks, which are available in larger sizes, are easier to handle, and enable rapid media exchange. For example, in BOBA culture, 10 mL of BME can be cultured in a single 225 cm 2 flask, and media exchange can be as simple as replacing the supernatant with a serum pipette. However, using dome methods, an equivalent culture would require nine 24-well plates (200 wells of 50 µL domes), and would require individual media exchange for each well. This also reduces plastic consumption by more than 70% (225 cm 2Flasks contain 152.7 g of plastic and replace 562.5 g of plastic in nine 24-well plates, data not shown). Further scale-up can be achieved using multi-layer flasks or “cell factories” that can hold several liters of culture volume. Table 1 provides general guidelines for the disclosed suspended BME hydrogel culture setup for several vessel types. It is reported that culture vessel type influences parameters such as gas transfer (Allen et al. (2001) Am J Physiol-Lung C 281, L1021-L1027), and factors such as media formulation, hydrogel composition, and organoid strain-specific growth rates can all influence organoid growth.

[0401] Table 1

[0402]

[0403] Seeding conditions for suspended BME hydrogel cultures.

[0404] The BOBA method also overcomes culture heterogeneity arising from hydrogel diffusion limitations in dome cultures (Park et al. (2022); Shin et al. (2020)). Some protocols suggest plating smaller 10-15 pL hydrogel domes (Pleguezuelos-Manzano et al. (2020); Stewart et al. (2020) Methods Mol Biology 2121, 185-198), thereby reducing the diffusion path for molecular transport. However, even when multiple domes are plated per well, plating smaller domes reduces the total hydrogel volume per well. Another method to overcome hydrogel diffusion limitations is to invert the plate during the hydrogel solidification step, causing gravity to cause cells to settle near the surface at the top of the dome, with few or no cells in the core of the dome (Pleguezuelos-Manzano et al. (2020)). This results in suboptimal use of expensive hydrogel volume and ultimately fewer organoid cells per pL of hydrogel that can be seeded. Complex bioengineering methods have been developed to increase the surface area of the hydrogel to improve molecular transport (Park et al. (2022)), but these are difficult to scale and still rely on anchoring the hydrogel to a 2D surface. The BOBA method produces more homogenous cultures without sacrificing the amount of hydrogel per well or the number of cells per pL of hydrogel. Without being limited to a particular theory, the observed improvement in organoid culture homogeneity can be explained by the BOBA hydrogel having (1) a smaller diameter and thus shorter diffusion path, and (2) all external surfaces exposed to the culture medium, enabling uniform diffusion of molecules into the hydrogel.

[0405] Another documented challenge of organoid morphological heterogeneity in hydrogel domes is that organoids located near the bottom of the plate can attach to the plate surface, spread out, and flatten, losing their 3D structure (Pleguezuelos-Manzano et al. (2020); Price et al. (2022)). Since the suspended hydrogel droplets do not come into direct contact with the plate surface, organoid spreading and flattening does not occur.

[0406] In addition to BOBA hydrogel droplets, organoids can also be grown as SOBA threads and SOBA thread fragments. Organoid growth is similar in all three configurations, demonstrating the robustness of the suspended BME culture method. The SOBA method is advantageous and speeds up culture preparation, as large volumes of cell-BME solution can be loaded into a single syringe to produce SOBA hydrogel threads. About 10 mL of SOBA can be produced in one minute, while equivalent dome cultures require over 15 minutes for plating and an additional 15-30 minutes for solidification. Despite the much greater length of SOBA compared to BOBA, no heterogeneity in organoid growth was observed (as seen in dome cultures), likely because the smaller diameter of SOBA (typically less than 2 mm) and enables efficient nutrient transport from the media. SOBA fragments are much closer in size and geometry to BOBA, but are produced much more quickly. Like BOBA, SOBA fragments are uniformly dispersed throughout the culture, which can be useful for kinetic sampling or dividing the culture for multiple applications or readouts.

[0407] Overall, the suspended hydrogel culture methods enable large-scale organoid scaling, improve organoid culture uniformity, and save labor, time, and resources. These culture improvements make intestinal organoids more suitable for high-throughput applications such as compound or genome screening. The methods can be extended to culture diseased intestinal organoids and organoid tumors, intestinal organoids from other species, and organoids from different tissue types. Thus, BOBA, SOBA, and SOBA threads have the potential to drive and expand the adoption of organoid technology.

[0408] Example 2: Use of Organoids in Screening Methods

[0409] This example discloses the use of intestinal organoids produced by the method of Example 1 in a cytotoxicity assay.

[0410] Methods

[0411] 96-well plate suspended BME organoid cytotoxicity assay. 225 cm 2Homogenized colon organoids SOBA fragments in flasks were seeded at 90 pL per well into two 96-well plates. Compound stocks were diluted to 10X final concentration in IntesticultOGM and 10 pL was added to each well. An 8-dose dilution series starting at 100 pM was technically evaluated in quadruplicate. After 3 days of treatment, viability measurements were performed using the Cell Titer Glo 3D assay kit (Promega) and luminescence was measured on an Ensight plate reader (Perkin Elmer). Prism (GraphPad) was used to generate 4PL fit curves.

[0412] Results:

[0413] Application of suspended BME hydrogel organoids in drug toxicity screening assays. Intestinal organoids produced in the form of BOBA, SOBA, or SOBA fragments can be used directly in downstream assays without additional organoid digestion or passaging steps. As a proof of concept, these organoids were demonstrated in drug toxicity screening. SOBA fragment-cultured organoids were grown in 96-well plates at 225 cm2per well and treated with known compounds that cause intestinal toxicity (diacerein, sorafenib, SN-38, or docetaxel) or DMSO vehicle control for 3 days. Viability was determined as a measure of ATP and dose response curves were generated (Figure 7E). 2 Growth in flasks (Figures 7A-7B), homogenized by trituration with a serum pipette, and then transferred to 96-well plates (Figures 7A-7C). The interwell variability, measured by Cell Titer Glo 3D ATP-based viability assay, was comparable for SOBA thread-grown organoids and dome cultures plated in 96-well plates (Figure 7D).

[0414] For drug toxicity screening, SOBA thread-grown organoids in 96-well plates were treated with known compounds that cause intestinal toxicity (diacerein, sorafenib, SN-38, or docetaxel) or DMSO vehicle control for 3 days. Viability was determined as a measure of ATP and dose response curves were generated (Figure 7E). Figure 7E This is one example of how suspended BME hydrogel cultures can be used directly for downstream applications.

[0415] The utility of suspended BME hydrogel growth cultures is demonstrated here in two applications. First, SOBA thread cultures can be homogenized to produce 96-well plate cultures with low interwell organoid variability. As a proof of concept, drug toxicity screening was performed; this method can be used for other medium-high throughput screens.

[0416] Example 3: Generation of Organoid-Derived Models

[0417] This example discloses the use of intestinal organoids produced by the method of Example 1 in providing an alternative organoid-derived model.

[0418] Methods

[0419] Transwell monolayers. 225 cm 2 Colon organoid SOBA fragments in flasks were collected into 50-mL conical tubes and pelleted at 800 x g for 3 minutes. Supernatant was removed and organoids were digested into single cells by incubation in TrypLE Express at 37°C water bath for 10 minutes, followed by trituration with a P1000 pipette. Incubation and trituration were repeated up to 2 times as needed. Cells were pelleted and washed with PBS, then resuspended in monolayer growth medium (Intesticult OGM + 10 µM Y-27632) or monolayer differentiation medium (Intesticult ODM + 10 µM Y-27632). For each well of a 96-well PET Transwell plate (pore size 0.4 µm, Corning catalog number 3450), 2.0 x 10 5 Cells were seeded into the top chamber and 200 µL of medium was added to the base chamber. Medium in both chambers was changed once every 2 to 3 days. Brightfield imaging was performed using a 10X objective and a THUNDER microscope (Leica) with a DFC9000 GTC camera (Leica). Trans epithelial electrical resistance (TEER) was determined by measuring electrical resistance with a volt / ohmmeter (EVOM3, WPI), then multiplying the electrical resistance by the Transwell surface area (0.143 cm 2 ).

[0420] Results:

[0421] Suspension BME hydrogel cultures facilitate alternative organoid-derived models. In addition to using organoids directly in their suspension BME hydrogel form, these organoids can also facilitate the generation of other organoid-derived models, especially those that require large cell inputs, such as monolayers and microphysiological system (MPS) devices. Suspension BME hydrogel organoids were used to generate colon epithelial Transwell monolayers and assess epithelial barrier function under two conditions. 225 cm 2SOBA spheroids grown in flasks were digested to single cells and plated on 96-well Transwell inserts at confluence and cultured for 7 days in either monolayer growth or monolayer differentiation media (Figure 8A). These two conditions produced Transwell epithelial cultures with different morphologies: in growth media, the resulting epithelium formed 3D structures protruding from the surface of the monolayer, whereas in differentiation media, these structures were absent and a typical“cobblestone” cell morphology of differentiated epithelial cells with mature tight junctions was visible (Figure 8B). Consistent with this morphology, by day 3, Transwell monolayers in differentiation media had higher epithelial barrier function, as quantified by measurement of transepithelial electrical resistance (TEER), than those in growth media Figure 8C This experiment is an example of how organoids grown using the BOBA, SOBA, or SOBA spheroid method can generate in vitro models that replace intestinal organoid-derived sources.

[0422] SOBA spheroid organoids can be used to generate organoid-derived Transwell monolayers, which offer the advantage of simultaneous access to apical and basolateral sides, but are difficult to scale up because they require large numbers of cells to seed. By enabling large-scale organoid expansion, the suspended BME hydrogel culture method can facilitate the development and implementation of complex intestinal organoid-derived model with higher tissue fidelity and experimental advantages.

[0423] Example 4: Generation of Lung Organoids

[0424] This example reveals the generation of lung organoids using the BME suspension method described herein.

[0425] Methods

[0426] Human lung ATII cell isolation and organoid derivation. De-identified human lung tissue from deceased donors was obtained through the Donor Network West. Lung dissociation into single cells and organoid establishment was previously described (Konishi et al., 2022). Briefly, tissue was washed with HBSS buffer and digested with digestion buffer (collagenase type I: 450 units / mL; dispase: 5 units / mL; DNase I: 10 units / mL). After removal of pleura and small airways, the remaining tissue was chopped with a single blade razor, transferred to a standard tube containing warm digestion buffer, and incubated at 37°C for a total of 1 hour with stirring and vigorous mixing every 15 minutes. The solution was passed through a 100 pm cell strainer and pelleted at 450 g for 10 minutes at 4°C. The cell pellet was resuspended in 5 mL ACK buffer to lyse red blood cells and incubated at room temperature for 3 to 5 minutes. The reaction was terminated by the addition of DMEM / F12 + 10% FBS. The cell suspension was then filtered through a 40 pm cell strainer and pelleted at 450 g for 5 minutes at 4°C. Cells were then labeled with CD45 magnetic microbeads (Miltenyi Biotech, catalog number 130-045-801) and loaded onto a MACS® column, which was placed in the magnetic field of a MACS Separator (according to the manufacturer’s protocol). Magnetically labeled CD45+ cells were retained in the column and discarded, and unlabelled cells were collected that passed through the column. These cells were washed in Gentle MACS buffer, pelleted at 450 g for 5 minutes at 4°C, and incubated with DMEM + 10% FBS containing 50 nM Lysotracker Green (Invitrogen, catalog number L7526) for 30 minutes at 37°C. The following antibodies were added to the cells on ice for 30 minutes: pre-conjugated HTII280 + Alexa 647 (Terrance Biotech; Invitrogen catalog number A20186); Epcam PE-Cy7 (BioLegend, catalog number 324222); CD45-Alexa700 (BioLegend, catalog number 304024), CD31-Alexa594 (BioLegend, catalog number 303126). The live / dead stain probe Sytox Blue (Invitrogen) was also added.The cell pellet was then washed, resuspended in PBS + 2% FBS, and ATII cells were isolated by sorting for Sytox blue-, CD45-, CD31-, EpCAM+, HTII-280+, lysotracker green+. ATII cells were resuspended in MATRIGEL® (Growth Factor Reduced, Phenol Red-Free; Corning, Cat# 356231) plated in 50 pL domes in 6-well plates (60 x 10 4 cells / mL, or 3000 cells / 50 pL MATRIGEL®), solidified for 30 minutes at 37°C, and then overlaid with 3 mL serum-free feeder-free (SFFF) media (Konishi et al., 2022) containing 10 pM ROCK inhibitor Y27632 (Selleckchem, Cat# S1049) for 3 days. After the first 2-3 days in culture, SFFF media without ROCK inhibitor was changed every 2-3 days.

[0427] Organoid maintenance. Organoid cultures were passaged every 10-14 days. To passage, cultures were first incubated with Accutase (Cell Stem Cell Technologies, Cat# 07922) for 15 minutes to soften the MATRIGEL®. Domes and organoids were collected in 15 mL tubes and incubated for 15 minutes at 37°C with agitation. After centrifugation at 450 g for 5 minutes at 4°C, cells were treated with TrypLE Express (Gibco, Cat# 12604-021) for 10 minutes at 37°C. TrypLE Express was inactivated by dilution with PBS and cells were pelleted at 450 x g for 5 minutes at 4°C. Cells were resuspended in ice-cold MATRIGEL® at 60 x 10 4 cells / mL, or 3000 cells / 50 pL MATRIGEL®), solidified for 30 minutes at 37°C. Domes were overlaid with SFFF media containing 10 pM ROCK inhibitor for the first 3 days, and then SFFF media without ROCK inhibitor was added and changed every 2-3 days.

[0428] Suspension of hydrogel BOBA cultures. Single organoid cells in MATRIGEL® were prepared on ice as described above. Pre-warmed SFFF medium containing 10 µM ROCK inhibitor was added to a 6-well plate (4 mL / well) and kept on a 37°C bath. To generate suspended BME droplets or BOBAs, organoid cells-MATRIGEL® solution was dispensed directly into the warm medium using an electronic serial pipette with a wide bore or cut pipette tip (opening ~2 mm) in 10 µL volumes. During dispensing, the tip was immediately immersed below the liquid surface and then lifted after each dispensing to ensure droplet detachment. After 3 days, SFFF medium was added and changed every 2 to 3 days. For medium exchange, a sterile 70 µm cell strainer was placed into the well, the dish was tilted and 3 mL of medium was gently sucked out through the strainer.

[0429] Results:

[0430] As shown in FIG. 9, lung alveolar type II (ATII) stem cells isolated from lung tissue embedded in a hydrogel suspended in culture medium were used to successfully generate lung organoids.

[0431] Example 5: Generation of Mammary Gland Organoids

[0432] This example provides a method of generating a breast organoid using the BME suspension method described herein.

[0433] Methods

[0434] Human breast organ...

Claims

1. A method for producing tissue-derived epithelial organoids, the method comprising: (a) Contacting tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture; (b) The hydrogel-tissue-derived epithelial stem cell mixture is suspended in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture; as well as (c) The suspended hydrogel-tissue-derived epithelial stem cell mixture is cultured in the culture medium to produce tissue-derived epithelial organoids.

2. The method of claim 1, wherein a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to produce the hydrogel-tissue-derived epithelial stem cell mixture.

3. The method of claim 2, wherein the plurality of tissue-derived epithelial stem cells comprise about 1 × 10⁻⁶ cells / year. 4 One tissue-derived epithelial stem cell / ml hydrogel to approximately 1 × 10⁻⁶ 7 One tissue-derived epithelial stem cell / ml hydrogel.

4. The method according to claim 2 or 3, wherein the plurality of tissue-derived epithelial stem cells are contained within tissue fragments, organoid fragments, or combinations thereof.

5. The method according to any one of claims 1 to 3, wherein the tissue-derived epithelial stem cells or the plurality of tissue-derived epithelial stem cells are isolated from primary epithelial tissue.

6. The method according to any one of claims 1 to 5, wherein the hydrogel solidifies upon contact with the culture medium.

7. The method according to any one of claims 1 to 6, wherein suspending the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium comprises immersing a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the culture medium.

8. The method according to any one of claims 1 to 7, wherein the temperature of the culture medium is from about 25°C to about 50°C.

9. The method according to any one of claims 1 to 8, wherein the temperature of the culture medium is from about 30°C to about 50°C.

10. The method according to any one of claims 1 to 9, wherein the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2°C to about 25°C.

11. The method according to any one of claims 1 to 10, wherein the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2°C to about 20°C.

12. The method according to any one of claims 1 to 11, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

13. The method of claim 12, wherein the natural hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

14. The method according to any one of claims 1 to 13, wherein the hydrogel has a storage modulus G' equal to or greater than the loss modulus G''.

15. The method according to any one of claims 1 to 14, wherein the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and / or diameter greater than about 0.1 mm.

16. The method of claim 15, wherein the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

17. The method according to any one of claims 1 to 16, wherein the hydrogel-tissue-derived epithelial stem cell mixture is suspended in droplets in the culture medium.

18. The method according to any one of claims 1 to 16, wherein the suspended hydrogel-tissue-derived epithelial stem cell mixture has a filamentous structure.

19. The method of claim 18, wherein the filamentous structure has a linear, serpentine, or spiral shape.

20. The method according to any one of claims 1 to 19, further comprising fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce a fragmented structure comprising the tissue-derived epithelial organoid.

21. A method for producing a suspension culture of tissue-derived epithelial organoids, the method comprising: (a) Introducing a mixture comprising hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspended mixture; as well as (b) The suspended mixture is cultured in the culture medium to produce epithelial organoids of the tissue source in a suspended state.

22. The method of claim 21, wherein the mixture introduced into the culture medium comprises the hydrogel and a plurality of tissue-derived epithelial stem cells.

23. The method of claim 22, wherein the plurality of tissue-derived epithelial stem cells comprise about 1 × 10⁻⁶ cells / year. 4 One tissue-derived epithelial stem cell / ml hydrogel to approximately 1 × 10⁻⁶ 7 One tissue-derived epithelial stem cell / ml hydrogel.

24. The method of claim 22 or 23, wherein the plurality of tissue-derived epithelial stem cells are contained within tissue fragments, organoid fragments, or combinations thereof.

25. The method according to any one of claims 21 to 23, wherein the tissue-derived epithelial stem cells or the plurality of tissue-derived epithelial stem cells are isolated from primary epithelial tissue.

26. The method according to any one of claims 21 to 25, wherein the hydrogel solidifies upon contact with the culture medium.

27. The method according to any one of claims 21 to 26, wherein introducing the mixture into the culture medium comprises immersing a dispensing device containing the mixture in the culture medium and dispensing the mixture into the culture medium.

28. The method according to any one of claims 21 to 27, wherein the temperature of the culture medium is from about 25°C to about 50°C.

29. The method according to any one of claims 21 to 28, wherein the temperature of the culture medium is from about 30°C to about 50°C.

30. The method according to any one of claims 21 to 29, wherein the temperature of the mixture is from about 2°C to about 25°C.

31. The method according to any one of claims 21 to 30, wherein the temperature of the mixture is from about 2°C to about 20°C.

32. The method according to any one of claims 21 to 31, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

33. The method of claim 32, wherein the natural hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

34. The method according to any one of claims 21 to 33, wherein the hydrogel has a storage modulus G' equal to or greater than the loss modulus G''.

35. The method according to any one of claims 21 to 34, wherein the suspended mixture has a geometry comprising a length, width and / or diameter greater than about 0.1 mm.

36. The method of claim 35, wherein the suspended mixture has a geometry comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

37. The method according to any one of claims 21 to 36, wherein the mixture is introduced into the culture medium in droplet form.

38. The method according to any one of claims 21 to 36, wherein the mixture is introduced into the culture medium in a filamentous structure.

39. The method of claim 38, wherein the filamentous structure has a linear, serpentine, or spiral shape.

40. The method according to any one of claims 21 to 39, further comprising fragmenting the suspended mixture to produce a fragmented structure comprising the tissue-derived epithelial organoid.

41. The method according to any one of claims 1 to 40, wherein the culture medium is contained in a container selected from the group consisting of: petri dishes, multi-well plates, conical tubes, reservoirs, culture bags, bioreactors, or flasks.

42. A method for producing a suspension culture of tissue-derived epithelial organoids, the method comprising: (a) Contacting tissue-derived epithelial stem cells with a hydrogel to produce a hydrogel-tissue-derived epithelial stem cell mixture; (b) Depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate; (c) Solidify the hydrogel-tissue-derived epithelial stem cell mixture to produce a solidified hydrogel-tissue-derived epithelial stem cell mixture; (d) The solidified hydrogel-tissue-derived epithelial stem cell mixture is suspended in a culture medium to produce a suspended hydrogel-tissue-derived epithelial stem cell mixture; as well as (e) The suspended hydrogel-tissue-derived epithelial stem cell mixture is cultured in the culture medium to produce tissue-derived epithelial organoids.

43. The method of claim 42, wherein a plurality of tissue-derived epithelial stem cells are contacted with the hydrogel to produce the hydrogel-tissue-derived epithelial stem cell mixture.

44. The method of claim 43, wherein the plurality of tissue-derived epithelial stem cells comprise about 1 × 10⁻⁶ cells / year. 4 One tissue-derived epithelial stem cell / ml hydrogel to approximately 1 × 10⁻⁶ 7 One tissue-derived epithelial stem cell / ml hydrogel.

45. The method of claim 43 or 44, wherein the plurality of tissue-derived epithelial stem cells are contained within tissue fragments, organoid fragments, or combinations thereof.

46. ​​The method according to any one of claims 42 to 44, wherein the tissue-derived epithelial stem cells or the plurality of tissue-derived epithelial stem cells are isolated from primary epithelial tissue.

47. The method according to any one of claims 32 to 46, further comprising removing the cured hydrogel-tissue-derived epithelial stem cell mixture from the substrate before suspending the cured hydrogel-tissue-derived epithelial stem cell mixture in the culture medium.

48. The method according to any one of claims 42 to 47, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

49. The method of claim 48, wherein the natural hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

50. The method according to any one of claims 42 to 49, wherein the hydrogel has a storage modulus G' equal to or greater than the loss modulus G''.

51. The method according to any one of claims 42 to 50, wherein the cured hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and / or diameter greater than about 0.1 mm.

52. The method of claim 51, wherein the cured hydrogel-tissue-derived epithelial stem cell mixture has a geometry comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

53. The method according to any one of claims 42 to 52, wherein the hydrogel-tissue-derived epithelial stem cell mixture is deposited as droplets onto the substrate.

54. The method according to any one of claims 42 to 52, wherein the hydrogel-tissue-derived epithelial stem cell mixture is deposited on the substrate to have a filamentous structure.

55. The method of claim 54, wherein the filamentous structure has a linear, serpentine, or spiral shape.

56. The method according to any one of claims 42 to 55, further comprising fragmenting the hydrogel-tissue-derived epithelial stem cell mixture in the culture medium to produce a fragmented structure comprising the tissue-derived epithelial organoid.

57. The method according to any one of claims 1 to 56, wherein the tissue-derived epithelial organoid has a uniform morphology compared to a reference tissue-derived epithelial organoid, wherein the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate.

58. The method of claim 57, wherein the tissue-derived epithelial organoid has a uniform size.

59. The method of claim 58, wherein the average diameter of the tissue-derived epithelial organoids is more uniform than that of the reference tissue-derived epithelial organoids.

60. The method according to any one of claims 1 to 59, wherein stem cells and / or proliferation markers are expressed at higher levels in the population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

61. The method of claim 60, wherein the stem cells and / or proliferation markers are selected from the group consisting of: MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof.

62. The method according to any one of claims 1 to 61, wherein the differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

63. The method of claim 62, wherein the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

64. A tissue-derived epithelial organoid produced by the method according to any one of claims 1 to 63.

65. A composition comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded within a hydrogel suspended in the culture medium.

66. The composition of claim 65, wherein the hydrogel has a geometry comprising a length, width and / or diameter greater than about 0.1 mm.

67. The composition of claim 66, wherein the hydrogel has a geometry comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

68. The composition according to any one of claims 65 to 67, wherein the hydrogel is in the form of droplets.

69. The composition according to any one of claims 65 to 67, wherein the hydrogel has a filamentous structure.

70. The composition of claim 69, wherein the filamentous structure has a linear, serpentine, or spiral shape.

71. The composition according to any one of claims 65 to 70, wherein stem cells and / or proliferation markers are expressed at higher levels in the population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

72. The composition of claim 71, wherein the stem cells and / or proliferation markers are selected from the group consisting of: MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof.

73. The composition according to any one of claims 65 to 72, wherein the differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

74. The composition of claim 73, wherein the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

75. A method for screening pharmaceutical agents, the method comprising: (a) Contacting the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids according to claim 64, or the composition according to any one of claims 65 to 74, with the pharmaceutical agent; and (b) Analyze changes in the tissue-derived epithelial organoids or populations of the tissue-derived epithelial organoids that indicate the effectiveness, treatment and / or toxicity of the agent.

76. The method of claim 75, wherein the agent is exposed to the tissue-derived epithelial organoids or a population of the tissue-derived epithelial organoids for about 15 minutes to about 3 years.

77. The method according to claim 75 or 76, wherein the agent is a therapeutic agent.

78. The method of claim 77, wherein the therapeutic agent is a peptide-based therapeutic agent, a small molecule therapeutic agent, a cell therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof.

79. A method for performing genome screening, the method comprising: (a) Providing a group of tissue-derived epithelial organoids or tissue-derived epithelial organoids as claimed in claim 64 or a composition as claimed in any one of claims 65 to 74; (b) A mutation is generated in the genome of one or more cells of the epithelial organoid derived from the said tissue; and (c) Analyze changes in the population of epithelial organoids of the tissue or of the tissue associated with the mutation.

80. The method of claim 79, wherein the mutation is generated using a gene regulation system.

81. The method according to claim 80, wherein the gene regulation system is a gene editing system.

82. The method according to claim 81, wherein the gene editing system is a CRISPR system.

83. The method according to any one of claims 75 to 82, wherein the change is a change in property, the property being selected from the group consisting of: cell viability, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, inhibition of cell signaling pathways, enzymatic activity, barrier integrity, and combinations thereof.

84. A method for generating an epithelial cell model, the method comprising: (a) Providing a population of tissue-derived epithelial organoids or tissue-derived epithelial organoids as described in claim 64; (b) Digesting the tissue-derived epithelial organoids or a population of the tissue-derived epithelial organoids into single cells; and (c) The single cell is cultured in a culture medium to produce a cell monolayer.

85. The method of claim 84, wherein the single cell is cultured on a permeable cell culture insert.

86. The method according to claim 84 or 85, wherein the culture medium is a differentiation culture medium.

87. The method according to claim 84 or 85, wherein the culture medium is a cell growth or stem cell promotion culture medium.

88. A method for screening pharmaceutical agents, the method comprising: (a) Contacting the cell monolayer produced by the method according to any one of claims 84 to 87 with the pharmaceutical agent; as well as (b) Analyze changes in the cell monolayer that indicate the effectiveness, treatment and / or toxicity of the agent.

89. The method of claim 88, wherein the agent is contacted with the cell monolayer for about 15 minutes to about 3 years.

90. The method according to claim 88 or 89, wherein the agent is a therapeutic agent.

91. The method of claim 90, wherein the therapeutic agent is a peptide-based therapeutic agent, a small molecule therapeutic agent, a cell therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof.

92. A method for performing genome screening, the method comprising: (a) Providing a cell monolayer produced by the method according to any one of claims 84 to 87; (b) Inducing mutations in the genome of one or more cells in the cell monolayer; and (c) Analyze the changes in the cell monolayer associated with the mutation.

93. The method of claim 92, wherein the mutation is generated using a gene regulation system.

94. The method according to claim 93, wherein the gene regulation system is a gene editing system.

95. The method according to claim 94, wherein the gene editing system is a CRISPR system.

96. The method according to any one of claims 84 to 95, wherein the change is a change in property, the property being selected from the group consisting of: cell viability, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, inhibition of cell signaling pathways, enzymatic activity, barrier integrity, and combinations thereof.

97. The method according to any one of claims 1 to 63 and 75 to 96 or the composition according to any one of claims 65 to 74, wherein the tissue fragments are fragments derived from tissue selected from the group consisting of: lacrimal glands, tonsils, salivary glands, gastrointestinal tissue, thyroid gland, lungs, mammary glands, liver, bile ducts, stomach, kidneys, pancreas, endometrium, fallopian tubes, cervix, prostate, bladder, taste buds, ovaries, placenta, and combinations thereof.

98. The method according to any one of claims 1 to 63 and 75 to 96 or the composition according to any one of claims 65 to 74, wherein the tissue-derived epithelial stem cells are obtained from fragments of organoids, said organoids being selected from the group consisting of: lacrimal gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, mammary gland organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, cytotrophoblast organoids, and combinations thereof.

99. A system for culturing tissue-derived epithelial organoids, comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium.

100. The system of claim 99, wherein the tissue-derived epithelial organoid is an intestinal organoid.

101. The system of claim 99 or 100, wherein the hydrogel has a geometry including a length, width and / or diameter greater than about 0.1 mm.

102. The system according to any one of claims 99 to 101, wherein the hydrogel has a geometry comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

103. The system according to any one of claims 99 to 102, wherein the hydrogel is in the form of droplets.

104. The system according to any one of claims 99 to 102, wherein the hydrogel has a filamentous structure.

105. The system of claim 104, wherein the filamentous structure has a linear, serpentine, or spiral shape.

106. The system according to any one of claims 99 to 105, wherein stem cells and / or proliferation markers are expressed at higher levels in the population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

107. The system of claim 106, wherein the stem cells and / or proliferation markers are selected from the group consisting of: MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof.

108. The system according to any one of claims 99 to 107, wherein the differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

109. The system of claim 108, wherein the differentiation marker is selected from the group consisting of: keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, keratin 19 (KRT19), keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

110. The system according to any one of claims 99 to 109, wherein the tissue-derived epithelial organoids are selected from the group consisting of: lacrimal gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, mammary gland organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, cytotrophoblast organoids, and combinations thereof.

111. The system according to any one of claims 99 to 110, comprising one or more robotic and / or automated components for generating and / or culturing epithelial organoids derived from said tissue.

112. The system of claim 111, wherein the one or more robots and / or automated components include a liquid handling robot.

113. A system for performing the method according to any one of claims 1 to 63 and 75 to 98, comprising one or more robots and / or automated components.

114. The system of claim 113, wherein the one or more robots and / or automated components include a liquid handling robot.

115. The method according to any one of claims 1 to 63 and 75 to 98, wherein one or more of the steps of the method are performed by one or more robots and / or automated components.

116. The method of claim 115, wherein the one or more robots and / or automated components comprise liquid handling robots.

117. The tissue-derived epithelial organoids according to claim 64, wherein the tissue-derived epithelial organoids are selected from the group consisting of: lacrimal gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, mammary gland organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, cytotrophoblast organoids, and combinations thereof.

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